Fast 3 3 diaminobenzidine tablets

Slides containing sections of formalin fixed, paraffin embedded (FFPE) rat prostate tissue were steamed for 25 min (E. coli IHC) or 40 min (Ki-67, CD68) in high temperature target retrieval (HTTR) solution for antigen retrieval (Dako). Slides were then incubated with an anti-E. coli antibody (Virostat, Inc., product #1001) at a dilution of 1:2000, anti-CD68 antibody (Abcam ab125212) at 1:1000, or anti-Ki-67 antibody (Novocastra) at 1:1000 for 45 min at room temperature, followed by incubation with secondary antibody (PowerVision, Leica Microsystems) for 30 min at room temperature. Staining was visualized using 3,3’-Diamino-benzidine (Sigma FAST 3,3’-Diamino-benzidine tablets), and slides were counterstained with hematoxylin.

Every eighth section containing mPFC was stained with the biotinylated lectin Wisteria floribunda agglutinin (WFA, Sigma Aldrich, catalog L1516). This lectin reacts with the CSPGs and is commonly used as one of the broadest markers for PNNs (Giamanco et al., 2010 (link); Härtig et al., 1994 (link)). Staining began by rinsing the tissue in triplicate with a tris buffered saline solution (TBS), followed by a 30 minute incubation at room temperature in a blocking solution (1% H₂O₂, 20% normal goat serum [NGS], 1% bovine serum albumin [BSA] in TBS) to prevent endogenous peroxide activity. Following blocking, the tissue was incubated for 24 hours at 4°C in WFA (1:500 concentration), which was diluted in Tris-Triton goat (TTG) solution (2% NSG, 0.3% Triton X-100 in TBS). The following day, sections were rinsed twice with both TTG and TBS, followed by a 60 minute incubation in avidin-biotin complex (ABC, Vectastain ABC Kit, Vector Laboratories) and triplicate 5-minute TBS rinses. Finally, slices were placed into a diaminobenzidine (DAB) solution in dH₂O (Sigma-Aldrich Fast 3–3’ Diaminobenzidine Tablets) for 2 minutes and then rinsed thoroughly with TBS to prevent further DAB reactions. Brain slices were mounted on electrostatically charged slides and allowed to dry for 24 hours before coverslipping with Permount (Fig.2a).