Immuno block reagent

Immunohistochemical staining protocols were based on our previous study 41 (link). Briefly, paraffin sections were treated with 3% H₂O₂ for 10 minutes and incubated with Immuno-Block reagent (BioSB) for 30 minutes at room temperature, followed by incubation with the primary antibody specifically against Ki67 (Abcam) and secondary antibody. Immunoreactive signal was visualized by enhanced DAB kit (Abcam).

The procedures of IF and IHC staining were described in detail in our previous reports [24 (link), 27 (link), 29 (link), 30 (link)]. For IHC and IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 min and incubated with Immuno-Block reagent (BioSB) for 30 min at room temperature. Sections were then incubated with primary antibodies specifically against matrix metalloproteinase (MMP)-9 (1:200, Thermo Fisher), α-tubulin (1:500, Santa Cruz), myeloperoxidase (MPO) (1:200, Bio SB), androgen receptor (AR) (1:100, Santa Cruz), vimentin (1:500, Santa Cruz), and CD68 (1:200, Abcam), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of the testicular specimen from each rat were analyzed. For quantification, three randomly selected HPFs (× 200 or × 400 for IHC and IF studies) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was determined by summation of all numbers divided by 9.

The procedure for IF staining has been described in our previous reports.²⁹, ³⁰ For IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 minutes and incubated with Immuno‐Block reagent (BioSB) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against CD14 (1:100, Santa Cruz) and F4/80 (1:100, Santa Cruz), whereas sections incubated with the use of irrelevant antibodies served as controls. Three sections of brain specimen from each animal were analysed. For quantification, three random HPFs (400× for IF studies) were analysed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

The procedure and protocol for IF staining have been reported in our previous studies 23 (link), 24 (link), 27 (link), 28 (link). For IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 minutes and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against F4/80 (1:100, Santa Cruz Biotechnology), CD14 (1:200, Proteintech), γ-H2AX (1:500, Abcam), p53BP1(1:300, Novus Biologicals) glial fibrillary acidic protein (GFAP) (1:500, Dako), CD31 (1:100, Bio-Rad), von Willebrand factor (vWF) (1:500, Merck Millipore), stromal cell-derived growth factor (SDF)-1α (1:100, Santa Cruz Biotechnology) and CXCR4 (1:200, Abcam), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of kidney specimen from each rat were analyzed. For quantification, three random HPFs (200× or 400× for ICH and IF studies) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

The procedure and protocol for IF staining have been reported in our previous studies [17 (link)–19 (link)]. For IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 min and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies specifically against CD31 (1:100, Bio-Rad), von Willebrand factor (vWF) (1:200, Merck Millipore), NeuN (1:1000, Millipore, Billerica, MA, USA), stromal cell-derived factor [(SDF)-1α (1:100, Santa Cruz Biotechnology), vascular endothelial growth factor (VEGF) (1:400, Abcam), CXCR4 (1:200, Thermo Fisher Scientific), microglial cell (1:100, Abcam, Cambridge, UK), CD68 (1:100,Abcam, Cambridge, UK), and glial fibrillary acidic protein (GFAP) (1:500, Dako) at 4 °C overnight. Alexa Fluor488, Alexa Fluor568, or Alexa Fluor594-conjugated goat anti-mouse or rabbit IgG were used to localize signals. Sections were finally counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40).
Three brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields (HPFs; × 400 for IF study) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

The procedure and protocol of IF staining have been reported in details in our previous and recent reports [41 (link)–45 (link)]. For IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 minutes and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against CD31 (1:100, BIO-RAD, CA, USA), vWF (1:200, Millipore, Massachusetts, USA) and TUNEL assay for apoptotic nuclei (In Situ Cell Death Detection Kit POD, Roche, Basel, Switzerland), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of LV specimen from each rat were analyzed. For quantification, three randomly selected HPFs (400x for IF study) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

IF staining has been described in our previous reports [32 (link), 35 (link), 37 (link)]. For IHC and IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 minutes and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against CD68 (1:100, Abcam, Cambridge, MA, USA), CD14 (1:300, BioSS, Woburn, MA, USA), kidney injury molecule (KIM)-1 (1:500, R&D Systems), MIF-1 (1:100, Abcam, Cambridge, MA, USA), and Wilm's tumor suppressor gene (WT)-1 (1:1000, Abcam, Cambridge, MA, USA). Sections incubated with irrelevant antibodies served as controls. Three sections of kidney specimens were analyzed in each rat. For quantification, three randomly selected high power fields (HPFs; 400x for IHC and IF studies) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined across all nine HPFs.
An IHC-based scoring system was adopted for semi-quantitative analyses of podocin and WT-1 as percentage of positive cells in a blind fashion [Score of positively-stained cell for podocin and WT-1: 0 = no stain %; 1= <15%; 2 = 15∼25%; 3 = 25∼50%; 4 = 50∼75%; 5= >75%-100%/per HPF (400 x)].