Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain

Reviewer #1 (Public Review):

In this study, Lin et al developed a protocol termed MOCAT, to perform tissue clearing and labelling on large-scale FFPE mouse brain specimens. They have optimised protocols for dewaxing and adequate delipidation of FFPE tissues to enable deep immunolabelling, even for whole mouse brains. This was useful for the study of disease models such as in an astrocytoma model to evaluate spatial architecture of the tumour and its surrounding microenvironment. It was also used in a traumatic brain injury model to quantify changes in vasculature density and differences in monoaminergic innervation. They have also demonstrated the potential of multi-round immunolabelling using photobleaching, as well as expansion microscopy with FFPE samples using MOCAT.

Comments on revised version:

The revised manuscript by Lin et al is much improved with a more detailed methods description. There are only a few minor comments for the authors:

- The new figures provided in Supplementary figure 5 did demonstrate a good level of transparency for the mouse brain tissue. However, quite marked tissue expansion can be seen following antigen retrieval and RI matching and this should be mentioned in the manuscript.
- The authors have provided comparison between mouse and human brain samples in Figure S12. However, it is misleading to mention that the "fluorescent signals are comparable at varying depth" as the figure clearly showed a lack of continuous staining especially for SMI312 at 900um depth, and human brain tissue showed considerably increased background signal (likely due to endogenous lipofuscin which has autofluorescent properties).
- It is understandable the authors cannot provide the full detail of the RI matching reagent as it is a commercialised product. However, it may still be useful if they can quote the refractive index +/- pH of the solution.

Reviewer #2 (Public Review):

The manuscript details an investigation aimed at developing a protocol to render centimeter-scale formalin-fixed paraffin-embedded specimens optically transparent and suitable for deep immunolabeling. The authors evaluate various detergents and conditions for epitope retrieval such as acidic or basic buffers combined with high temperatures in entire mouse brains that had been paraffin-embedded for months. They use various protein targets to test active immunolabeling and light-sheet microscopy registration of such preparations to validate their protocol. The final procedure, called MOCAT pipeline, briefly involves 1% Tween 20 in citrate buffer, heated in a pressure cooker at 121 {degree sign}C for 10 minutes. The authors also note that part of the delipidation is achieved by the regular procedure.

Major Strengths
- The simplicity and ease of implementation of the proposed procedure using common laboratory reagents distinguish it favorably from more complex methods.

- Direct comparisons with existing protocols and exploration of alternative conditions enhance the robustness and practicality of the methodology.

Major Weaknesses

- The assertion that MOCAT can be rapidly applied in hospital pathology departments seems overstated due to the limited availability of light-sheet microscopes outside research labs. In the first rebuttal letter, authors explain the limitations of other microscopes more readily available in hospitals. This explanation relies on your own investigations and practical experience on the matter, so including them in some part of the manuscript would be beneficial.

- Refractive index matching is a critical point in the protocol, the one providing final transparency. Authors utilized the commercial solutions NFC1 and NFC2 (Nebulem, Taiwan) with a known refractive index, but for which its composition is non-disclosable. My knowledge on the organic chemistry around refractive index matching is limited, but if users don't really know what is going on in this final step, the whole protocol would rely on a single world-wide provider and troubleshooting would be fishing. I suggest that you try to validate the approach with solutions of known composition, or at least provide the solutions sold by other providers.

Final considerations
The evidence presented supports the effectiveness of the proposed method in rendering thick FFPE samples transparent and facilitating repeated rounds of immunolabeling.

The developed procedure holds promise for advancing tissue and 3D-specific determination of proteins of interest in various settings, including hospitals, basic research, and clinical labs, particularly benefiting neuroscience research.

The methodological findings suggest that MOCAT could have broader applications beyond FFPE samples, differentiating it from other tissue-clearing approaches in that the equipment and chemicals needed are broadly accessible.

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations for the Authors):

(1) Within the section on "optimized antigen retrieval", the authors mentioned that weak immunolabelling and strong non-specific labelling may be due to inadequate antigen retrieval. I wonder whether this interpretation is accurate. Could it also be due to inadequate antibody penetration?

We appreciate the reviewer's comment and have revised our text to improve clarity. Regarding the SDS-electrophoresed sample (Figure S1a right), we acknowledge that the brain-surrounding background noise indicates insufficient antibody penetration. However, in the FLASH-processed sample (Figure S1a left), the background signal is uniformly distributed throughout the entire brain. Therefore, we conclude that incomplete antibody penetration is unlikely under this condition. Below is the revised paragraph:

Revised manuscript, line 62-66: “We observed that both FLASH-processed and SDS-electrophoresed samples showed weak tyrosine hydroxylase (TH, a marker of dopaminergic neurons) signal (Figure S1a, Supporting Information). Additionally, we noticed that the FLASH-processed samples had almost no signal of NeuN, a marker of neuronal nuclei (Figure S1b left, Supporting Information), and exhibited strong non-specific background noise (Figure S1a left, Supporting Information). The presence of this background noise is considered an indicator of inadequate antigen retrieval.[48]”

• Also, the authors mentioned the use of FLASH protocol and SDS-based electrophoresis for delipidation which were not described in the methods section.

We have included the information in the revised Materials and Methods.

Revised manuscript, line 418-426: S”HIELD processing, SDS-electrophoretic delipidation and FLASH delipidation. PFA-fixed specimens were incubated in SHIELD-OFF solution at 4 °C for 96 hours, followed by incubation for 24 hours in SHIELD-ON solution at 37 °C. All reagents were prepared using SHIELD kits (LifeCanvas Technologies, Seoul, South Korea) according to the manufacturer's instructions. For SDS-electrophoretic delipidation, SHIELD-processed specimens were placed in a stochastic electro-transport machine (SmartClear Pro II, LifeCanvas Technologies, Seoul, South Korea) running at a constant current of 1.2 A for 5-7 days. For FLASH delipidation, the SHIELD-processed specimens were placed in FLASH reagent (4% w/v SDS, 200 mM borate) and then incubated at 54 ℃ for 18 hours.[47] The delipidated specimens were washed with PBST at room temperature for at least 1 day.”

• In addition, tyrosine hydroxylase (TH) should be a marker of "monoaminergic" neurons rather than specifically "dopaminergic" neurons.

We appreciate the reviewer's correction. It is true that tyrosine hydroxylase (TH) is a marker for neurons that contain dopamine, norepinephrine, and epinephrine (catecholamines). However, the adrenergic and noradrenergic neurons are relatively few and are mostly located in the medulla and brain stem. Since we only monitoring the brain in this study, we wish to keep TH as an indicator of dopaminergic neurons.

(2) It was mentioned that tissue integrity was retained following heating treatment during the MOCAT protocol. It would be useful to demonstrate any differences in structural distortion, if any, with before and after images with different delipidation agents.

We have provided an additional supplementary figure (Figure S5 in the revised manuscript) to display the mouse brain at different stages of the MOCAT protocol, including pre-delipidation, post-delipidation, and post-RI-matching, to demonstrate the tissue integrity.

Revised manuscript, line 135-137: “Figure S5 shows the gross views of the same mouse brain after undergoing 4% PFA fixation, paraffin processing, optimized antigen retrieval, and RI-matching, demonstrating intactness of the brain shape and preservation of tissue integrity.”

(3) In this study, the authors have demonstrated the protocol could be successfully applied to FFPE specimens up to 15 years old. However, archival brain bank materials often have brain tissues with extended formalin fixation time. It may be useful to demonstrate that this technique can be utilised on FFPE tissues with long formalin fixation times.

We appreciate the reviewer's suggestions. We have included an additional supplementary figure (Figure S6) to demonstrate the application of MOCAT to 3-month fixed mouse brain hemispheres. Although the long-term fixed specimens exhibited reduced TH intensity and S/N ratio, the major dopaminergic regions were labeled, and magnified images revealed details of cell bodies and neuronal fibers. These results suggest that MOCAT has the potential to be applied to long-term fixed specimens.

The fluorescence intensity was more affected by fixation with formalin, which is methanol-stabilized and stronger, than with PFA. This indicates that a stronger antigen retrieval method may be a possible solution. However, achieving the right balance between antigen retrieval efficiency and tissue integrity will require additional testing and investigation.

Revised manuscript, line 163 to 167: “We also applied MOCAT to 3-month fixed mouse brain hemispheres (Figure S6). Although the long-term fixed specimens exhibited reduced TH intensity and S/N ratio, the major dopaminergic regions were labeled, and magnified images revealed clear details of cell bodies and neuronal fibers. These results suggest that MOCAT has the potential to be applied to long-term fixed specimens.”

Revised manuscript, line 346-351: “In the demonstration of MOCAT to 3-month fixed specimens, we observed that pontine reticular nucleus (Figure S6A, yellow arrowheads) lose TH-positive signals after long-term fixation. The fluorescence intensity was more affected by fixation with formalin, which is methanol-stabilized and stronger, than with PFA. The results indicate that a stronger antigen retrieval method may be a possible solution. However, achieving the right balance between antigen retrieval efficiency and tissue integrity will require additional testing and investigation.”

(4) Whilst it is encouraging to see this protocol enables multi-round immunolabelling, further work is required to demonstrate there is no cross-reactivity in subsequent rounds of immunostaining following bleaching (e.g. Non-specific secondary antibody binding).

We appreciate the reviewer for noting their concern and providing suggestions. To address this issue, we have examined the results of the second to fourth rounds of multi-round staining, as shown in Figure 3. In all three sequential rounds, we utilized rabbit primary antibodies and the same secondary antibodies. Our observations under a 3.6x objective (NA = 0.2) did not reveal any colocalization with the staining from the previous round. Hence, we conclude that cross-reactivity is not significant. However, we acknowledge the need for more comprehensive testing to completely rule out the possibility of cross-reactivity, such as employing antibodies from different hosts or utilizing different types of secondary antibodies (e.g., IgG, Fab2).

Revised manuscript line 189-191: “The brain shape and structural integrity remained after 4 rounds of immunolabeling, and there is no cross-reactivity in subsequent rounds of immunostaining following bleaching. (Figure S11).”

• Also, how was the structural integrity maintained for tissues after multiple rounds of heat-induced epitope retrieval?

We have provided an additional supplementary figure (Figure S11 in the revised manuscript) to demonstrate the structural integrity after 4 rounds of immunolabeling.

Revised manuscript line 189-191: “The brain shape and structural integrity remained after 4 rounds of immunolabeling, and there is no cross-reactivity in subsequent rounds of immunostaining following bleaching (Figure S11).”

(5) It may be useful to have a side-by-side comparison in staining quality with equivalent sizes of rodent and human brain tissues as there appeared to be a reduction in clarity and staining quality at greater imaging depth for human tissues.

We have provided an additional supplementary figure (Figure S12) to show the fluorescent images of TH- and Lectin-labeling in 1mm-thick human and mouse brain tissues at depths of 100 um, 500 um, and 900 um. For millimeter-sized samples, both human and mouse brains showed comparable levels of transparency, with no noticeable reduction in fluorescence signal at varying depths. In our forthcoming studies, we plan to conduct a more comprehensive comparison of centimeter-sized human and mouse brain tissues.

(6) Lectin staining is used throughout this study to label vasculature of the brain. How specific is this as compared with other vasculature markers such as CD31?

We appreciate the reviewer for addressing their concern. Lectins are nonimmune-origin carbohydrate-binding proteins that have been utilized to label the surface of the blood vessel lumen. On the other hand, CD31, CD34, etc. are immunomarkers of vascular endothelial cells. Numerous references have confirmed that lectin staining consistently co-localizes with CD31 immunoreactivity (Battistella et al. 2021; Miyawaki et al. 2020). However, in tumors, blood vessels lacking a lumen may display CD31 positive/Lectin negative conditions (Morikawa et al. 2002).

(7) When discussing the applicability of MOCAT on the astrocytoma mouse model, there is a bit of confusion with regard to the terminology. As astrocytoma by default will be comprised of astrocytes, it may be useful to describe the tumour astrocytes as ASTS1CI-GFP positive astrocytes and immunolabelled astrocytes as GFAP-positive astrocytes.

We thank the reviewer for their suggestions. To avoid confusion for readers, we have made modifications to the content and labeling of Figure 6A.

Revised manuscript, line 213-219: “…we subjected an intact FFPE brain from an astrocytoma mouse model (see Materials and Methods) to the MOCAT pipeline to label tumor cells (ASTS1CI-GFP positive astrocytes) and GFAP-positive astrocytes (Figure 6A, C). Accordingly, we could segment GFAP-positive astrocytes surrounding the tumor (Figure 6B, D, and E) and classify them according to their distances from the tumor cells. Statistical analysis (Figure 6F) revealed that nearly half of the GFAP-positive astrocytes were within the tumor, with 63.9% being located near the tumor surface (±200 μm).”

(8) Within the methods section, further details of the antibodies such as the clonality and immunogen should be included in the supplementary table.

We appreciate the reviewer for their suggestions. In the revised version, we have included these details in Supplementary Table 1.

• Furthermore, there is inadequate detail regarding multi-round immunolabelling and the precise timing of immunolabelling including lectin staining, various imaging parameters including the working distance of the lens and excitation laser used.

We have added the experimental details of multi-round staining for Figure 3 in Supplementary Table 3. This table now includes information about the amounts and types of chemicals and antibodies used, as well as the laser wavelengths used for each round. The staining conditions (including labeling time, temperature, and buffer used) have been disclosed in Materials and Methods (see MOCAT pipeline/Electrophoretic immunolabeling). Furthermore, we have included the working distance and NA value of the objective lens used in MOCAT pipeline/Volumetric imaging and 3D visualization subsection.

Revised manuscript, line 464-479: “Electrophoretic immunolabeling (active staining). The procedure was modified from the previously published eFLASH protocol[15] and was conducted in a SmartLabel System (LifeCanvas Technologies, Seoul, South Korea). The specimens were preincubated overnight at room temperature in sample buffer (240 mM Tris, 160 mM CAPS, 20% w/v D-sorbitol, 0.9% w/v sodium deoxycholate). Each preincubated specimen was placed in a sample cup (provided by the manufacturer with the SmartLabel System) containing primary, corresponding secondary antibodies and lectin diluted in 8 mL of sample buffer. Information on antibodies, lectin and their optimized quantities is detailed in Supplementary Table 1. The specimens in the sample cup and 500 mL of labeling buffer (240 mM Tris, 160 mM CAPS, 20% w/v D-sorbitol, 0.2% w/v sodium deoxycholate) were loaded into the SmartLabel System. The device was operated at a constant voltage of 90 V with a current limit of 400 mA. After 18 hours of electrophoresis, 300 mL of booster solution (20% w/v D-sorbitol, 60 mM boric acid) was added, and electrophoresis continued for 4 hours. During the labeling, the temperature inside the device was kept at 25 ℃. Labeled specimens were washed twice (3 hours per wash) with PTwH (1× PBS with 0.2% w/v Tween-20 and 10 μg/mL heparin),[23] and then post-fixed with 4% PFA at room temperature for 1 day. Post-fixed specimens were washed twice (3 hours per wash) with PBST to remove any residual PFA.”

Revised manuscript, line 483-490: “Volumetric imaging and 3D visualization. For centimeter-scale specimens, images were acquired using a light-sheet microscope (SmartSPIM, LifeCanvas Technologies, Seoul, South Korea) with a 3.6x customized immersion objective (NA = 0.2, working distance = 1.2 cm). For samples <3 mm thick, imaging was performed using a multipoint confocal microscope (Andor Dragonfly 200, Oxford Instruments, UK) with objectives that were UMPLFLN10XW (10x, NA = 0.3, working distance = 3.5 mm), UMPLFLN20XW (20x, NA = 0.5, working distance = 3.5 mm), UMPLFLN40XW (40x, NA = 0.8, working distance = 3.3 mm). 3D visualization was performed using Imaris software (Imaris 9.5.0, Bitplane, Belfast, UK).”

• Also, since refractive index homogenisation is an important step in tissue-clearing experiments, it may be useful to describe the components of NFC1 and NFC2 solutions used and provide images of the "cleared" tissues.

We have included the image of a cleared mouse brain in Figure S5. Additionally, we have provided the refraction index of NFC1 and NFC2 in Materials and Methods (see MOCAT pipeline/Refractive index matching). However, the composition of NFC1 and NFC2, being commercialized products from Nebulem (Taiwan), is non-disclosable.

Reviewer #2 (Public Review):

Major Weaknesses:

• There is no evidence of actual transparency of the entire mouse brain across different treatments. The suggested protocol is very good at removing lipids (as assessed by DiD staining) and by results of fluorescence registration deep within the brain. BUT, since in many places of the manuscript authors speak of "transparency" the reader will expect the typical picture in which control and processed brains are on top of a white graphical pattern that would evidence transparency (see as an example Figure 1 and 2 of Wan et al. 2018 (Neurophotonics. 2018 Jul;5(3):035007. doi: 10.1117/1.NPh.5.3.035007.)

We thank the reviewer for their suggestions. We have provided an additional supplementary figure (Figure S5 in the revised manuscript) to demonstrate the transparency.

• The manuscript lacks clarity on the applicability of MOCAT to regular formalin-fixed tissue and tissues other than the brain.

We appreciate the reviewer's suggestions. We have included an additional supplementary figure (Figure S6) to demonstrate the application of MOCAT to a 3-month regular formalin-fixed mouse brain hemisphere. We observed that the major dopaminergic regions were still labeled, although with reduced intensity and S/N ratio. We also observed that the fluorescence intensity was more affected in formalin, which is methanol-stabilized and stronger, than in PFA, implying that a stronger antigen retrieval method may be possible to rescue the intensity. However, achieving the right balance between antigen retrieval efficiency and tissue integrity will require additional testing and investigation.

Revised manuscript, line 163 to 167: “We also applied MOCAT to 3-month fixed mouse brain hemispheres (Figure S6). Although the long-term fixed specimens exhibited reduced TH intensity and S/N ratio, the major dopaminergic regions were labeled, and magnified images revealed clear details of cell bodies and neuronal fibers. These results suggest that MOCAT has the potential to be applied to long-term fixed specimens.”

Revised manuscript, line 346-351: “In the demonstration of MOCAT to 3-month fixed specimens, we observed that pontine reticular nucleus (Figure S6A, yellow arrowheads) lose TH-positive signals after long-term fixation. The fluorescence intensity was more affected by fixation with formalin, which is methanol-stabilized and stronger, than with PFA. The results indicate that a stronger antigen retrieval method may be a possible solution. However, achieving the right balance between antigen retrieval efficiency and tissue integrity will require additional testing and investigation.”

Regular formalin

We agree with the reviewer and plan to investigate the potential use of MOCAT in tissues other than the brain in our subsequent studies.

• Insufficient information is provided on the "epoxy treatment" or "hydrogel," and a more detailed explanation is warranted.

We appreciate the reviewer's question. In response, we have included a paragraph in the Discussion section to clarify the appropriate timing for using epoxy or hydrogel in the MOCAT pipeline. However, the harsh conditions, such as pressure and heat, caused by external forces might damage specimens. To protect specimens from the harsh conditions caused by active staining, specimens could be strengthened by treatment with epoxy or acrylamide monomer to form a tissue-epoxy or tissue-hydrogel hybrid.[29,31] Laboratories that do not have adequate devices or handle small specimens could use passive immunolabeling instead and skip the step of epoxy or hydrogel pretreatment.

Epoxy and acrylamide hydrogel can both strengthen tissue structures. However, in this study, we only used epoxy for treatment in combination with active electrophoretic staining. To avoid confusion and improve clarity, we have made modifications to Figure 1B and included epoxy processing in the MOCAT pipeline subsection within Materials and Methods.

Revised manuscript, line 329-340: “In Figure 1B, we propose two staining strategies for samples with thicknesses less than 500 um and greater than 1 mm: passive immunolabeling and active immunolabeling. In passive immunolabeling, antibodies penetrate and reach their targets solely through diffusion, without any additional force. It takes approximately two months to passively stain a whole mouse brain.[26,28] Compared to passive immunolabeling, active immunolabeling uses an external force, such as pressure, electrophoresis, etc., to facilitate antibody penetration and therefore significantly speed up the staining process, reducing the required staining time for a whole mouse brain to one day. However, the harsh conditions, such as pressure and heat, caused by external forces might damage specimens. To protect specimens from the harsh conditions caused by active staining, specimens could be strengthened by treatment with epoxy or acrylamide monomer to form a tissue-epoxy or tissue-hydrogel hybrid.[29,31] Laboratories that do not have adequate devices or handle small specimens could use passive immunolabeling instead and skip the step of epoxy or hydrogel pretreatment.”

• The differences between passive and active immunolabeling, as well as photobleaching data, should be addressed for a comprehensive understanding.

We appreciate the reviewer's question. We have included a paragraph in the Discussion section to explain the differences between passive and active immunolabeling:

Revised manuscript, line 329-340: “In Figure 1B, we propose two staining strategies for samples with thicknesses less than 500 um and greater than 1 mm: passive immunolabeling and active immunolabeling. In passive immunolabeling, antibodies penetrate and reach their targets solely through diffusion, without any additional force. It takes approximately two months to passively stain a whole mouse brain.[26,28] Compared to passive immunolabeling, active immunolabeling uses an external force, such as pressure, electrophoresis, etc., to facilitate antibody penetration and therefore significantly speed up the staining process, reducing the required staining time for a whole mouse brain to one day.”

Regarding the effects of photobleaching, we have added Figure S10 to demonstrate the efficiency of using our approach.

Revised manuscript, line 184-185: After imaging, we photobleached transparent RI-matched samples using a 100W LED white light to quench the previously labeled fluorophores (Figure S10).

• The assertion that MOCAT can be rapidly applied in hospital pathology departments seems overstated due to the limited availability of light-sheet microscopes outside research labs.

We thank the reviewer's question. Since the imaging depth primarily relies on the working distance of the objective lens, if a long working distance objective lens (such as UMPLFLN10XW from Olympys Inc.) is available, it is also possible to scan samples up to a thickness of approximately 3.5mm. However, confocal systems require longer scanning times, and in non-optical sectioning wide-field fluorescence microscopes like the Olympus BX series or ZEISS Axio imager series, deconvolution algorithms must be utilized to eliminate out-of-focus signals.

Additionally, the epifluorescence system may also result in reduced fluorescent intensity in the deeper regions of the sample. If the fluorescent signal of the target is weak or exceeds the working distance of the objective lens, an alternative option is to send the sample to a microscopy or imaging facility core for scanning and further analysis.

• The compatibility of MOCAT with genetically encoded fluorescent proteins remains unclear and warrants further investigation.

We appreciate the reviewer's question. We have included a paragraph in the Discussion section to address this limitation of MOCAT:

Revised manuscript, line 354-361: “Fourth, MOCAT is not compatible with endogenous fluorescence due to a reduction in fluorescence intensity caused by xylene and alcohol used in paraffin processing. Researchers who need to directly observe genetically encoded fluorescent proteins can utilize tissue-clearing methods such as 3DISCO, X-CLARITY, CUBIC, etc., which have been shown to minimize the decrease in fluorescence intensity. On the other hand, if researchers need to visualize transgenic fluorescent proteins along with other biomarkers, they can use MOCAT for delipidation and boost-immunolabeling to visualize the transgenic fluorescent proteins.”

• The control of equivalent depths in cryosections for evaluating the intensity of DiD staining should be elaborated upon.

We have included these information in the section of Materials and Methods:

Revised manuscript, line 428-430: “Serial 20-µm-thick cryosections were cut from mouse brain slices (2-mm thick) of various treatment conditions for subsequent DiD or Oil red O staining. For DiD staining, cryosections (that were of approximately 0-40 µm depth) were post-fixed with 4% PFA at room temperature for 5 minutes.”

• The composition of NFC1 and NFC2 solutions for refractive index matching should be provided.

We have provided the refraction index of NFC1 and NFC2 in Materials and Methods (see MOCAT pipeline/Refractive index matching). However, the composition of NFC1 and NFC2, being commercialized products from Nebulem (Taiwan), is non-disclosable.

Reviewer #2 (Recommendations for the Authors):

• A larger readership would benefit from validating imaging depths using fluorescence microscopies commonly found in pathological departments (i.e. Confocal, 2-photon, epifluorescence+deconvolution, etc).

We thank the reviewer's recommentation. Since the imaging depth primarily relies on the working distance of the objective lens, if a long working distance objective lens (such as UMPLFLN10XW from Olympys Inc.) is available, it is also possible to scan samples up to a thickness of approximately 3.5mm. However, confocal systems require longer scanning times, and in non-optical sectioning wide-field fluorescence microscopes like the Olympus BX series or ZEISS Axio imager series, deconvolution algorithms must be utilized to eliminate out-of-focus signals.

Additionally, the epifluorescence system may also result in reduced fluorescent intensity in the deeper regions of the sample. If the fluorescent signal of the target is weak or exceeds the working distance of the objective lens, an alternative option is to send the sample to a microscopy or imaging facility core for scanning and further analysis.

-Investigate the compatibility of MOCAT with genetically encoded fluorescent proteins, a common target in research specimens.

We appreciate the reviewer's question. We have included a paragraph in the Discussion section to address this limitation of MOCAT:

Revised manuscript, line 354-361: “Fourth, MOCAT is not compatible with endogenous fluorescence due to a reduction in fluorescence intensity caused by xylene and alcohol used in paraffin processing. Researchers who need to directly observe genetically encoded fluorescent proteins can utilize tissue-clearing methods such as 3DISCO, X-CLARITY, CUBIC, etc., which have been shown to minimize the decrease in fluorescence intensity. On the other hand, if researchers need to visualize transgenic fluorescent proteins along with other biomarkers, they can use MOCAT for delipidation and boost-immunolabeling to visualize the transgenic fluorescent proteins.” References:

Battistella, Roberta et al. 2021. “Not All Lectins Are Equally Suitable for Labeling Rodent Vasculature.” International Journal of Molecular Sciences 22(21): 22. /pmc/articles/PMC8584019/ (January23, 2024).

Miyawaki, Takeyuki et al. 2020. “Visualization and Molecular Characterization of Whole-Brain Vascular Networks with Capillary Resolution.” Nature Communications 2020 11:1 11(1): 1–11. https://www.nature.com/articles/s41467-020-14786-z (January23, 2024).

Morikawa, Shunichi et al. 2002. “Abnormalities in Pericytes on Blood Vessels and Endothelial Sprouts in Tumors.” The American Journal of Pathology 160(3): 985–1000.