Journal: bioRxiv
Article Title: Deep transcriptome profiling of human hypothalamic agouti-related protein and proopiomelanocortin neurons regulating energy homeostasis
doi: 10.1101/2025.08.19.671088
Figure Lengend Snippet: A : Schematic overview of histological procedures used to evaluate the combined effects of postmortem delay and immersion fixation (24, 48, or 72 h) on RNA integrity. B : Neocortical tissue samples from a 53-year-old female, processed in the presence of the RNase inhibitor aluminon (ATA; 0.05%), show comparable RNA quality (RIN > 7.0) and yield across different fixation times (Agilent Bioanalyzer; Table S1). C : Experimental design for optimizing RNA preservation using ATA (0.05%) or polyvinyl sulfonic acid (PVSA; 1-10%) during immunohistochemical detection of cortical parvalbumin (PVALB) neurons. D : Both ATA and PVSA provide significant protection against degradation during antigen retrieval at 80 °C, while RNA integrity is severely compromised in the absence of RNase inhibitors; ATA and PVSA (2%) also maintain RNA integrity during IHC performed either at RT or 4 °C (Table S1). E : Effects of ATA (0.05%) and PVSA (2%) on IHC signal quality and background. Both additives enable successful detection of PVALB neurons; however, ATA treatment results in increased nonspecific background staining. Statistical annotations: **p<0.01, ##p<0.00001, ###p<0.000001 by one-way ANOVA followed by Tukey’s HSD.
Article Snippet: Sections were pretreated with 1% H 2 O 2 and 0.5% Triton X-100 in PBS for 20 minutes, followed by incubation with a commercially available affinity-purified goat polyclonal antibody against human parvalbumin (#EB06776; Everest Biotech; 1:30,000) for either 40 hours at 4 °C or overnight at RT.
Techniques: Preserving, Immunohistochemical staining, Staining