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.

Strengths:
This paper has demonstrated, with some good imaging examples, that it is possible to perform deep immunostaining with detailed analysis on FFPE samples using MOCAT. The figures provided appeared to be largely convincing with good amount of details.

They have showcased different ways to perform analysis on cleared tissue. For example, the use of lectin-labelled blood vessels as a structural reference for multi-round immunolabelling was very useful. They have also demonstrated how to generate comparable quantitative data on various mouse disease models which will be important for future tissue-clearing studies.

Weaknesses:
Although the authors have proven the feasibility of their techniques on FFPE samples, it is questionable whether this will translate well for human brain tissues. The vast majority of the study data was generated using rodent brain tissues and it appears the technique was only performed on human FFPE tissues no larger than 1 mm in thickness. The PFA/formalin fixation time for the tissue was also limited to 24 hours in this study. Whilst this may be true for most surgical specimens, whole brain specimens in brain banks will often have formalin fixation time exceeding 3 weeks. The issue of prolonged formalin fixation prior to embedding in paraffin wax was not addressed in this study.

Inherent differences in human and rodent brain tissues may affect the effectiveness of immunostaining. In this study, results on human brain specimens appeared to show a reduction in clarity and staining quality at greater imaging depth at 900 µm, particularly for MAP2 and GFAP (Figure 5).

In addition, there are inadequate details in the materials and methods section which may limit the readers' ability to successfully replicate the study or proposed method for tissue clearing. Further details on the optimisation of this protocol and brief details from previously published protocols were not described in the methods section.

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
- 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.)

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

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

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

- 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.

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

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

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

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

We appreciate your comments and also thanks to the reviewers for providing valuable feedback and recommendations. For most of the recommendations, we will respond in the revised version, which will provide more information for readers to understand and apply the study. For some of the recommendations, we can give quick responses as follows:

Reviewer #2 (Public Review):

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

In passive immunolabeling, antibodies penetrate and achieve their targets merely via diffusion, without any additional force. In contrast, active immunolabeling utilizes an external force, such as pressure, electrophoresis, etc., to facilitate antibody penetration and therefore significantly speed up the staining process (i.e., one day vs. 2 months for a whole mouse brain). In our study, the samples we were dealing with were centimeter-sized; therefore, we employed only active electrophoretic immunolabeling (details provided in Materials and Methods). However, for laboratories that do not possess adequate devices or handle small specimens, they can employ passive immunolabeling instead. As for the photobleaching data, we will provide it in the revised version.

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

We agree with the possibility that the encoded fluorescent proteins will be affected. Since there is evidence that fluorescence can be quenched by xylene and alcohol, which are two organic solvents used in paraffin processing, we think boost immunolabeling is necessary for observing genetically encoded fluorescent proteins. We also pointed out this limitation in the Discussion:

“Fourth, endogenous fluorescence—such as GFP, YFP, and tdTomato—may be quenched during paraffin processing and thus need to be visualized by means of additional immunolabeling.”

However, the extent to which endogenous fluorescence will be quenched during the paraffin processing and MOCAT procedure, and how much boost labeling can rescue, is worth investigating for broadening the application of MOCAT. We will provide it in the revised version.

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

Since NFC1 and NFC2 are commercial products from Nebulem (Taiwan), the composition is non-disclosable. However, the refractive index of NFC1 and NFC2 is 1.47 and 1.52, respectively.