Immpress hrp horse anti mouse igg polymer detection kit

For Masson’s Trichrome’s staining, kidney tissues were fixed for 48 h at RT in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks, and sectioned at 4 μm. Sections were then stained with Masson’s Trichrome according to the standard protocol and examined by light microscopy. For immunohistochemistry, kidneys were processed as mentioned above. Following dewaxing and antigen retrieval processes (98 °C for 20 min in citrate buffer), kidney sections were permeabilized with 0.1% Triton X-100 (Sigma), incubated with 3% H₂O₂ (Sigma), and blocked with 2.5% normal horse serum (S-2012, Vector Labs). Kidney sections were incubated with primary antibody (X203 and X209 at 1:250 dilution in PBST) overnight (4 °C) and visualized using an ImmPRESS HRP horse anti-mouse IgG polymer detection kit (MP-7402, Vector Labs) with ImmPACT DAB Peroxidase Substrate (SK-4105, Vector Labs). Hematoxylin (H-3401, Vector Labs) was used to stain the nuclei prior to imaging by light microscopy.

Processing for histological evaluation involved excision of material implant and sham sites along with a margin of surrounding normal tissue. Tissue explants were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) or Masson's Trichrome (MTC). For immunohistochemistry, sections were deparaffinized, rehydrated, processed for antigen retrieval, and stained with mouse anti-rat CD68 antibody (pan macrophage marker; 1:100; Bio-Rad, Herceles, CA, USA), rabbit anti-rat CD3 (T cell marker; 1:1000; Abcam, Waltham, MA, USA), rabbit anti-rat CD4 (T helper cell marker; 1:1200; Abcam), mouse anti-rat CD8a (cytotoxic T cell marker; 1:200; ThermoFisher Scientific, Waltham, MA, USA), rabbit anti-rat Foxp3 (Treg marker; 1:300; Abcam), or mouse anti-rat IL-17 (Th17 marker; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Slides were then treated with ImmPRESS® HRP Horse Anti-Mouse IgG Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) or ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories) and counterstained with hematoxylin. Histological assessments were performed by an independent pathologist in a blinded fashion.