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Cryopreservation of aldehyde-fixed whole brains.

Code ↔ Paper

2 matches between paragraphs of the paper and lines of its authors' code, computed by the harvester (lexical-v1). Click a colored paragraph or line to see its counterpart.

The 2 matches
  1. [1] § Materials and methods › Histological methods ↔ cryo_em_data_analysis.R, lines 1–55 · score 0.70 · Mann Whitney, Hodges Lehmann shift, cryopreserved control, probability, AUC, rank
  2. [2] § Results › Histological assessment of the refined protocol ↔ cryo_em_data_analysis.R, lines 1–55 · score 0.66 · Mann Whitney, Hodges Lehmann shift, cryopreserved control, bootstrap, AUC, rank

Paper

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The authors' code

R · 97 lines · 3.7 KB · no license · 2 matches

  1. library(irr)
  2. library(ggplot2)
  4. df <- read.delim("Cryo\ blinding\ -\ org\ sheet.tsv", sep = "\t", header = TRUE)
  6. # Fix column names
  7. colnames(df) <- c("sample", "image", "number", "m_score", "a_score")
  9. # Note: "N" = Non-Cryopreserved control, "C" = "Cryopreserved"
  11. # Fill down sample IDs (they're only on first row of each sample)
  12. current_sample <- ""
  13. for (i in 1:nrow(df)) {
  14. if (df$sample[i] != "" && !is.na(df$sample[i])) {
  15. current_sample <- df$sample[i]
  16. } else {
  17. df$sample[i] <- current_sample
  18. }
  19. }
  21. # Extract group (c vs n) from sample name
  22. df$group <- ifelse(grepl("^c", df$sample), "c", "n")
  24. # Average raters, then aggregate to sample level
  25. df$combined <- rowMeans(df[c("m_score", "a_score")])
  26. sample_means <- aggregate(combined ~ sample + group, data = df, FUN = mean)
  28. cat("=== Group Comparison ===\n")
  29. aggregate(combined ~ group, data = sample_means,
  30. FUN = function(x) c(n = length(x), mean = round(mean(x), 2), median = round(median(x), 2)))
  32. wt <- wilcox.test(combined ~ group, data = sample_means, conf.int = TRUE)
  33. print(wt)
  34. cat("Hodges-Lehmann shift (c - n):", round(wt$estimate, 3), "\n")
  35. cat("95% CI:", round(wt$conf.int[1], 3), "to", round(wt$conf.int[2], 3), "\n")
  37. # AUC (Mann-Whitney probability of superiority) with bootstrap 95% CI
  38. x <- sample_means$combined[sample_means$group == "c"]
  39. y <- sample_means$combined[sample_means$group == "n"]
  40. n1 <- length(x); n2 <- length(y)
  41. AUC <- as.numeric(wt$statistic) / (n1 * n2)
  43. set.seed(1)
  44. B <- 10000
  45. aucs <- numeric(B)
  46. for (b in 1:B) {
  47. xb <- sample(x, replace = TRUE)
  48. yb <- sample(y, replace = TRUE)
  49. ranks <- rank(c(xb, yb))
  50. Ub <- sum(ranks[1:n1]) - n1 * (n1 + 1) / 2
  51. aucs[b] <- Ub / (n1 * n2)
  52. }
  53. ci_auc <- quantile(aucs, c(0.025, 0.975))
  54. cat("AUC (P[cryo > control]):", round(AUC, 3), "\n")
  55. cat("Bootstrap 95% CI:", round(ci_auc[1], 3), "to", round(ci_auc[2], 3), "\n")
  57. # Create ratings matrix (each row = one image, columns = raters)
  58. ratings_m_a <- cbind(df$m_score, df$a_score)
  60. cat("=== Interrater Reliability (M vs A) ===\n")
  61. cat("N images rated:", nrow(ratings_m_a), "\n\n")
  63. # ICC - two-way random, absolute agreement, single measures
  64. icc_result <- icc(ratings_m_a, model = "twoway", type = "agreement", unit = "single")
  66. cat("ICC:", round(icc_result$value, 3), "\n")
  67. cat("95% CI:", round(icc_result$lbound, 3), "to", round(icc_result$ubound, 3), "\n")
  69. # (i) Image and sample N per condition and region
  70. df$region <- ifelse(as.numeric(gsub("[cn]", "", df$sample)) <= 9, "gm", "wm")
  71. sample_means$region <- ifelse(as.numeric(gsub("[cn]", "", sample_means$sample)) <= 9, "gm", "wm")
  73. cat("=== Image counts by condition x region ===\n")
  74. print(table(df$group, df$region))
  75. cat("\n=== Sample counts by condition x region ===\n")
  76. print(table(sample_means$group, sample_means$region))
  78. # distribution plot
  80. df$region <- ifelse(as.numeric(gsub("[cn]", "", df$sample)) <= 9, "Grey matter", "White matter")
  81. df$group_label <- ifelse(df$group == "c", "Cryopreserved", "Control")
  82. df$sample <- factor(df$sample, levels = c("c7","c8","c9","n7","n8","n9","c10","c11","c12","n10","n11","n12"))
  84. p <- ggplot(df, aes(x = sample, y = combined, color = group_label)) +
  85. geom_jitter(width = 0.15, height = 0.05, alpha = 0.7, size = 2) +
  86. stat_summary(fun = median, geom = "crossbar", width = 0.5, color = "black", linewidth = 0.4) +
  87. facet_grid(. ~ region, scales = "free_x", space = "free_x") +
  88. scale_y_continuous(breaks = 1:5, limits = c(0.5, 5.5)) +
  89. scale_color_manual(values = c("Cryopreserved" = "#1f77b4", "Control" = "#d62728")) +
  90. labs(x = "Specimen", y = "Mean rater score per image (1 = best, 5 = worst)",
  91. color = NULL) +
  92. theme_bw() +
  93. theme(legend.position = "top",
  94. panel.grid.minor = element_blank())
  96. print(p)

cryo_em_data_analysis.R at commit 3f8b354, no license · at the source

Overview

Authors: Macy Garrood1, Alicia Keberle1, Andria Slaughter1, Allison Sowa2, Emma L Thorn3,4, Claudia De Sanctis3,4, Kurt Farrell3,4, John F Crary3,4, Andrew T McKenzie1
ORCID iDs: Andrew T McKenzie
  1. Apex Neuroscience, Salem, Oregon, United States of America
  2. Microscopy and Advanced Bioimaging Core, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
  3. Friedman Brain Institute, Departments of Pathology, Neuroscience, and Artificial Intelligence & Human Health, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
  4. Neuropathology Brain Bank & Research Core and Ronald M. Loeb Center for Alzheimer’s Disease, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America
Institutions: Icahn School of Medicine at Mount Sinai (United States)
Journal: PloS one, volume 21, issue 8, article e0344932
Dates: received 5 March 2026; accepted 1 August 2026; published online 24 August 2026
Type: Research article · Language: English
License: CC BY
Identifiers: DOI 10.1371/journal.pone.0344932 · PMID 42636208 · PMCID PMC13502592 · OpenAlex W7204133711
Open access: gold, a free copy (OpenAlex)
Status: code verified
Categories: human (organism)
Methods: Statistics
MeSH: Aldehydes*, Brain*, Cryopreservation*, Tissue Fixation*, Cryoprotective Agents, Humans (* major topic)
Topic: Molecular Biology Techniques and Applications (Molecular Biology, Biochemistry, Genetics and Molecular Biology), according to OpenAlex
Funding: NIA NIH HHS (K01 AG070326, P30 AG066514, RF1 AG062348); NIMH NIH HHS (RF1 MH128969); NINDS NIH HHS (RF1 NS095252, U54 NS115266)
Citations: not cited yet (Europe PMC); 43 references in the paper
Research resources: RRID:SCR_009969, RRID:SCR_027565

Abstract

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules, motivating interest in alternative long-term preservation strategies, such as cryopreservation. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for preserving fixed whole brains using graded immersion cryoprotection followed by subzero temperature storage. We refer to this general strategy – aldehyde fixation followed by cryoprotectant loading and subzero storage – as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose in fixative. We used CT imaging to track cryoprotectant penetration, finding that approximately 9 months is required for the CT signal to stabilize throughout whole human brains. In our initial validation experiment, insufficient equilibration time prior to freezing led to ice crystal artifacts in the white matter. After refining the protocol to allow adequate diffusion time, light and electron microscopy showed preserved cellular architecture and ultrastructure. Our approach may be valuable for laboratories seeking a method for long-term subzero storage of fixed whole brain specimens.

Reproduced under the paper's license (CC BY), from the paper cited above.

Repository

Its files are read in the Code ↔ Paper reader above, with 2 matches between paragraphs and lines of code.

andymckenzie/Aldehyde_based_cryopreservation

License: none: the authors keep all their rights
State: the link answers, verified on 27 September 2026
Evidence: files inventoried
Commit: 3f8b354fb869e2e617995c1ee25656e914d45f36, 2 June 2026
Languages: R (1)
Size: 2 files, 1 script
Software Heritage: not archived
Found in: “Data Availability”
Not found: README, license file, CITATION.cff, environment file, tests, continuous integration, documentation
Tools: ggplot2 (1 file)
Availability: 1 check, the latest on 27 September 2026: the link answers
  • 27 September 2026: the link answers
1 file
At the source: github.com/andymckenzie/Aldehyde_based_cryopreservation

The paper's code and data availability statement is in the Data section.

Tracing map

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  • 1 repository of the authors' code, each at its verified commit, with its license and how the link was found in the paper;
  • 1 script, each with its path and the digest of its content;
  • 2 matches between paragraphs of the paper and lines of the code (method lexical-v1);
  • neither the text of the paper nor the code itself.

Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.

Data

Datasets cited

Data Availability

Whole slide image and electron microscopy data can be accessed in a public community on Zenodo (https://zenodo.org/communities/aldehyde_based_cryopreservation). The specific datasets are available at the following DOIs: 10.5281/zenodo.20514794, 10.5281/zenodo.18805394, 10.5281/zenodo.18601441, and 10.5281/zenodo.18601466. Code and data used for data analysis are available from GitHub (https://github.com/andymckenzie/Aldehyde_based_cryopreservation).

Reproduced under the paper's license (CC BY), from the paper cited above.

Versions

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Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 9 authors, 6 MeSH terms, 3 funders, 41 references, 2 RRIDs.

Cite

This paper

Garrood, M., Keberle, A., Slaughter, A., Sowa, A., Thorn, E. L., De Sanctis, C., Farrell, K., Crary, J. F., & McKenzie, A. T. (2026). Cryopreservation of aldehyde-fixed whole brains. PloS one, 21(8), e0344932. https://doi.org/10.1371/journal.pone.0344932

BibTeX

@article{garrood2026cryopreservation,
author = {Garrood, Macy and Keberle, Alicia and Slaughter, Andria and Sowa, Allison and Thorn, Emma L and De Sanctis, Claudia and Farrell, Kurt and Crary, John F and McKenzie, Andrew T},
title = {{Cryopreservation of aldehyde-fixed whole brains}},
journal = {PloS one},
year = {2026},
month = aug,
volume = {21},
number = {8},
pages = {e0344932},
publisher = {PLOS},
issn = {1932-6203},
doi = {10.1371/journal.pone.0344932},
url = {https://doi.org/10.1371/journal.pone.0344932},
pmid = {42636208},
pmcid = {PMC13502592}
}

RIS

TY - JOUR
AU - Garrood, Macy
AU - Keberle, Alicia
AU - Slaughter, Andria
AU - Sowa, Allison
AU - Thorn, Emma L
AU - De Sanctis, Claudia
AU - Farrell, Kurt
AU - Crary, John F
AU - McKenzie, Andrew T
TI - Cryopreservation of aldehyde-fixed whole brains
T2 - PloS one
J2 - PLoS One
PY - 2026
DA - 2026/08/24
VL - 21
IS - 8
SP - e0344932
SN - 1932-6203
PB - PLOS
DO - 10.1371/journal.pone.0344932
UR - https://doi.org/10.1371/journal.pone.0344932
LA - en
ER -

CSL-JSON

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