Immpress hrp horse anti rabbit igg

Kidney tissue samples were fixed with 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and cut into 5-μM sections. For immunohistochemistry staining, tissue sections were heated in an antigen retrieval buffer. Then, the sections were incubated with 3% hydrogen peroxide, followed by a blocking buffer. The slides were then exposed to the primary antibody at 4 °C overnight. After washing, the slides were incubated with ImmPRESS HRP Horse Anti-Rabbit IgG (MP-7401; Vector Laboratories) or ImmPRESS-AP Horse Anti-Goat IgG (MP-5405; Vector Laboratories) for 1 hour at room temperature. After washing, color was developed with an ImmPACT DAB Substrate Kit, Peroxidase (HRP) (SK-4105; Vector Laboratories) and ImmPACT Vector Red Substrate Kit, Alkaline Phosphatase (AP) (SK-5105; Vector Laboratories). The primary antibodies for immunostaining included antimacrophage (ab22506, Abcam) and antie-α-SMA (ab5694, Abcam). Sirius red staining was performed according to a standard protocol from the manufacturer (Chondrex Inc).

Tumor bearing omenta from young and aged mice (n=13) [3 (link)] were fixed with 10% formalin, then paraffin embedded and sectioned (5μm). Sections were de-paraffinized by xylene and were rehydrated in decreasing concentrations of ethanol. Antigen retrieval was done with 10mM sodium citrate, pH 6.0 (SMA, AGE antibodies) or Tris-EDTA buffer (10mM Tris, 1mM EDTA, 0.05% Tween 20, pH 9.0; DPP4 antibody). Endogenous peroxidases were blocked with 3% H₂O₂. Slides were blocked in horse serum, then incubated overnight at 4°C with an anti-AGE antibody (Abcam, ab23722, 1:10,000), an anti-SMA antibody (Abcam, ab5694, 1:1000), or anti-DPP4 antibody (Abcam, ab187048, 1:20,000) diluted in horse serum. All antibodies were then incubated with ImmPRESS HRP Horse Anti-Rabbit IgG (Vector Labs MP-7401) according to manufacturer’s specifications, developed with 3,3-diaminobenzene and counterstained with hematoxylin. Tissues were dehydrated in increasing concentrations of ethanol followed by xylene and mounted with Cytoseal XYL. Slides were imaged with Aperio ScanScope CS and analyzed with ImageScope software.