PONE-D-21-05943

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

PLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript to PLOS ONE. I would like to apologize for how long it has taken to provide you with reviews.  After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The major concerns of the reviewers concerned your statistical analysis and the quality of the images:  You should use ANOVA followed by a post hoc test for multiple comparisons, rather than t tests.  As far as the images are concerned, the reviewers may have been interpreting the pdf versions, rather than the high definition tiff images that can be downloaded from the website.  However, reviewer 2 points out that fluorescence microscopy and immunofluorescence (IF) staining of rodent orbits with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunofluorescence staining with this method,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Li and colleagues present a study comparing eight cryo-histological protocols for both quality and repeatability. The article is well written, the methods are clearly described, and the data will be beneficial for the scientific community. Though the paper is directed towards identifying the BTA method as both the most reproducible and highest quality, the authors present images of tissue from all eight methods in the supplemental data. Perhaps this figure could be moved to the main part of the manuscript, as it is central to the hypothesis tested in this study. These qualitative impressions would pair nicely with the quantitative data in Figure 3. My only concern of any significance is that it would appear that a student-t test was done in which the score from each technique was compared to the BTA technique. If the experimental design was to prepare tissue with eight different methods, evaluate the sections and identify which method produced the best result, a student-t test would not be the correct statistical tool. Perhaps a one-way ANOVA with a post-hoc Tukey-Kramer multiple comparisons test would be more appropriate?

Specific Comments:

Figure 1, 4, 5: The image quality in Figure 3 was acceptable. The image quality in Figure 1 is so poor I cannot evaluate the tissue. The image quality in 4, 5, and 6 is better than Figure 1, but still not great. Please increase the resolution. The images in the Supplemental Materials were of high quality.

Figure 2: This is extremely helpful and a wonderful visual aid towards providing the reader with a visual explanation of the experimental design.

Figure 3A: BTA – would the scale bar appear more clear in black? Perhaps this is why it not aligned with the other scale bars.

Figure 3B: Please label the y-axis. If 12 was the largest value (perfect score), why does the graph go to 14?

Supplemental Table 3: Are the quality scores presented an average? If so, please provide the SEM.

The data points behind the means were not provided in this study, which I believe is required (Figure 3/Table2/Table S3).

Additional comments:

Line 68 there is an extra space between “easy” and “collection”

Reviewer #2: The manuscript entitled “Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs” describes a new frozen sectioning method adapted to keep the morphology of rodent orbit morphology intact. The authors systematically evaluate the outcomes of routine H&E-stained sections with this new method compared with those obtained with other histology methods.

Major concerns relate to insufficient validation of the proposed approach.

1. In the eye research field, immunohistochemical staining techniques with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunoperoxidase stained with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape-aided sectioning, staining, and aqueous mounting medium).

2. In the eye research field, fluorescence microscopy and immunofluorescence (IF) staining of rodent orbits with cell-specific and molecular markers are critical. The authors should assess the morphological outcomes of immunofluorescence staining with this new method, and compare TSA (Tape aided sectioning, staining, and aqueous mounting medium) and WSA (without tape aided sectioning, staining, and aqueous mounting medium). If IF is affected, the results should be disclosed with alternate techniques.

3. The authors should determine whether the use of 15% EDTA for 21 days for decalcification of the whole head specimens affects the immunostains. The use optimizes the concentration and duration of immersion with EDTA for immunostaining.

4. Lines 8 and Line 42, "OCT (optimal compound of tissue)" should be "OCT (optimal cutting temperature) compound".

5. Line 213, To compare the quality of section staining, only the best 1 out of the 10 serial sections at the position Bregma+3.90mm was selected for evaluation. They should perform the analysis of the sections at 5 different positions (Bregma +2.60, +3.25, +3.90, +4.55-, and +5.20-mm) as the serial quality of the sectioning is critical for 3D reconstruction.

6. The optical resolution of all the figures is suboptimal photos with better resolution should be provided.

7. The authors should disclose the rate of section loss and the rate of tissue folding. Table 2 showed the success rate for each of the 8 groups. If more than half of the stained section area is damaged or covered by folded tissue, it should be counted as section loss.

8. The authors should add a video as supplementary information.

9. Line 38. The authors should change “…serial whole integrated sections

of orbits or eyeballs to determine further precise site-specific morphological changes that respond to treatments or genetic modifications.” to “…serial whole sections

of orbits or eyeballs to determine morphological changes that respond to treatments or genetic modifications.”

10. Line 43. The tissue thickness should be disclosed.

11. Line 113. The authors should disclose the 3 dimensions of the tissue blocks.

12. Line 117: High concentration of saturated picric acid represents a biosafety risk. An alternate solution should be provided.

13. Line 140, The dimensions of the slide of glass and supplier should be disclosed.

14. Line 170 The authors should write the full name of BTA here.

15. Line 213. The authors state that “One best quality section of the right-side orbital tissues among the ten for each head was selected for section quality comparisons among the various protocols according to staining section quality.” The authors should justify, and explain whether the selection process was performed in a masked fashion.

16. They should indicate whether the person who performed the sectioning was different or not.

17. Line 228: They should indicate the person(s) who assessed the sections with initials.

18. The authors should disclose freezing artifacts such as vacuolization at high power in soft tissue such as retina that can be noticed at high power (X 40 objective). They should also disclose the distribution of freezing artifacts. The authors should show high power images of the deeper structures such as the retina (X 40 objective).

19. The results section should be separated from the discussion section.

20. The results with tissue types other than eye and orbit tissues should be excluded from this manuscript, as the results are preliminary and the quality of the sections has not been validated.

21. Line 367: The authors should disclose the total duration of processing from sacrifice to sectioning.

22. In the limitations section the authors should include uneven HE staining that may prevent the use of these sections for morphometric studies.

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Reviewer #1: No

Reviewer #2: No

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Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

PONE-D-21-05943R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Alfred S Lewin, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the authors’ thought comments and responses to reviewer feedback. All significant concerns have been addressed. Of slight note, it appears there is a typo in the last sentence of the Abstract that I missed on the first review: Application of this protocol in joints, brains, and so on, - the challenging integral sectioning tissues, also generated high-quality histological staining sections.

Reviewer #2: The authors addressed all comments and queries.

Aminor suggestion:

Line 273 Bregman should be changed to Bregma.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No