post hoc immunostaining  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: HumanIslets: An integrated platform for human islet data access and analysis

doi: 10.1101/2024.06.19.599613

Figure Lengend Snippet: Mini-case study: Linking SERCA3b expression with diabetes and islet function. A) Analysis of proteomic data comparing the corrected diabetes status of ‘no diabetes’ versus ‘type 2 diabetes’. Analyses were run with no correction, with addition of age, sex and BMI as covariates, and then with addition of ‘proportion non-endocrine’ as a covariate. Adjusted p-value <0.05 indicated in red. B) Web-tool Results Table, with top hits sorted by adjusted p-value. C) Clicking on ‘SC’ (single-cell) raises a visualization of single-cell RNA-seq data for a particular hit (in this case ATP2A3 ) to confirm cell-type specific expression. D) Clicking on a row of the Results Table raises the protein expression of a hit (in this case ATP2A3) separated by the initial query. This example shows ATP2A3 expression in donors with no diabetes (ND), pre-type 2 diabetes (pre-T2D) and T2D (Type2). A potential outlier donor is indicated in the red circle. E) Clicking on the potential outlier raises the Donor View page for that donor for inspection. Shown here are the donor metadata, HbA1c and pancreas weight in relation to the total database donor population, and cell type composition for this potential outlier (R241). Composition calculations are from the proteomics deconvolution, with exocrine cells shown as a proportion of all cells and endocrine cell types shown as a proportion of all endocrine cells. Inset, the presence of both insulin (green) and glucagon (red) positive cells in R241 is confirmed by fluorescence immunostaining in banked FFPE biopsy from this donor. F) Proteomic data downloaded from the Data Download page, can be analyzed by the user. In this case the potential outlier was removed from analysis of ATP2A3 expression (****-p<0.0001; one-way ANOVA followed by Tukey post-test to compare groups). G) Gene set enrichment analysis, via the ‘pathway analysis’ tab highlights pathways up-regulated (blue) and down-regulated (red) in T2D. Results were exported in table format and plotted using local software. Within the tool, clicking on a pathway result raises a heatmap showing genes/proteins contributing to the pathway. Significant hits are indicated by ***. H) In the results table (panel B), clicking on ‘NCBI’ takes users to the NCBI gene page for a given hit, while clicking on ‘T2DKP’ takes users to the T2D Knowledge Portal page. Data extracted from the T2DKP page for top endocrine-enriched hits demonstrates Human Genetic Evidence (HuGE) scores suggesting potential links to human metabolism and diabetes. Red shows HuGE scores for ATP2A3 . I) In the results table (panel B), clicking on the hit name (in this case ATP2A3) takes users to the Feature View results for that particular hit. Here, ATP2A3 is seen to be negatively (red) associated with T2D at both the transcript and protein levels, and positively (blue) associated with some measures of beta-cell function and insulin secretion.

Article Snippet: Following electrophysiological measurements, cells were identified by post-hoc immunostaining for insulin with a rabbit anti-insulin primary antibody (Santa Cruz; #SC-9168; RRID: AB_2126540) and goat anti-rabbit Alexa Fluor488 secondary (ThermoFisher, #A-11076; RRID: AB_141930), and with a guinea pig anti-glucagon primary antibody (Sigma-Aldrich, #G2654; RRID: AB_259852) and goat anti-guinea pig Alexa Fluor 594 secondary (ThermoFisher, #A-11076; RRID: AB_141930); or following collection for scRNA-seq.

Techniques: Expressing, RNA Sequencing, Fluorescence, Immunostaining, Software, Cell Function Assay