dapi spectral counterstaining  (Akoya Biosciences)

Journal: Reproduction (Cambridge, England)

Article Title: A lack of commensal microbiota influences the male reproductive tract intergenerationally in mice

doi: 10.1530/REP-24-0204

Figure Lengend Snippet: Significant transcriptomic changes occur in the GF cauda associated with immune cells. (A, B) Venn diagrams depict the overlap of DEGs that are (A) upregulated and (B) downregulated in GF reproductive tissues compared to SPF mice. (C) Volcano plot illustrating the log 2 fold change ( x -axis) and log 10 adjusted P -value (P-adj; y -axis) of genes detected in the GF cauda compared to the SPF cauda. Dots highlighted orange and green indicate those genes that satisfy the criteria of fold change >±1.5 and P -adj ≤0.05 and therefore identified as significantly altered. (D) Bar plot indicating the distribution of gene location (top) and type (bottom) in all genes and DEGs in the GF cauda epididymis as determined by Ingenuity Pathway Analysis. (E, F) Expression data were input into the ImmunCellAI to infer the differences in immune cell populations between SPF and GF tissue based on gene expression of cell markers. (E) Abundance score for macrophages in SPF and GF cauda tissue and (F) Log 2 fold change of five macrophage cell markers in the GF cauda epididymis when compared to SPF as measured by RNA-seq. (G) Heatmap depicting the log 2 average TPM of SPF and GF cauda and log 2 TPM for replicate 4 of the GF cauda (GF4) from RNA-seq for 41 genes increased in the GF cauda epididymis and classified as transmembrane receptor. (H) Immunofluorescence staining of cauda epididymis with antibodies against the macrophage marker F4/80 (green) and co-stained with DAPI (blue) from SPF and GF mice. Scale bar = 200 μm. L – lumen and asterisks indicate infiltration of macrophages in the interstitium.

Article Snippet: Finally, sections were incubated in the Akoya Biosciences TSA reagents Opal 520 (OP-1001) for 10 min, followed by Spectral DAPI nuclear counterstain (Akoya Biosciences FP1490) and mounted with Fluoromount-G (SouthernBiotech, USA 100-01).

Techniques: Expressing, Gene Expression, RNA Sequencing, Immunofluorescence, Staining, Marker

Journal: Reproduction (Cambridge, England)

Article Title: A lack of commensal microbiota influences the male reproductive tract intergenerationally in mice

doi: 10.1530/REP-24-0204

Figure Lengend Snippet: Transcriptomic changes in F1 offspring emulate F0 changes. (A) Volcano plots depicting the log 2 fold change ( x -axis) and log 10 adjusted P -value (P-adj; y -axis) of genes detected in F1 GF × SPF (GxS) caput (left) and cauda (right) epididymis compared to SPF. Dots highlighted orange and green indicate genes that satisfied the criteria of >±1.5-fold change and P -adj ≤0.05 and therefore identified as significantly altered. (B) Venn diagram depicting the number of overlapping DEGs in the caput (left) and cauda (right) epididymis between GF F0 and F1 compared to SPF. (C) Correlation plot of the expression in GF vs SPF ( x -axis: log 2 fold change) compared with GxS F1 vs SPF ( y -axis: log 2 fold change) cauda epididymis. Blue colored dots indicate GxS F1 vs SPF DEGs, yellow dots indicate GF F0 vs SPF DEGs, while orange and green dots indicate up- and downregulated DEGs shared between GF and GxS F1 offspring when compared to controls. (D) Heatmap of the log 2 fold change of the top 10 up- and downregulated genes in the caput and cauda epididymis shared between GF and GxS F1 mice. (E) F4/80 (green) and DAPI (blue) immunofluorescent staining in the cauda epididymis of GxS F1 mice. Scale bar = 200 μm. L – lumen and asterisks indicate infiltration of macrophages in the interstitum.

Article Snippet: Finally, sections were incubated in the Akoya Biosciences TSA reagents Opal 520 (OP-1001) for 10 min, followed by Spectral DAPI nuclear counterstain (Akoya Biosciences FP1490) and mounted with Fluoromount-G (SouthernBiotech, USA 100-01).

Techniques: Expressing, Staining