Vector novared substrate kit for peroxidase

Sections were placed in 0.1 M phosphate-buffer saline solutions supplemented with 0.3% Triton X-100 and 2% normal goat serum, and then incubated for 48 h at 4°C with a rabbit polyclonal antibody against c-FOS (sc-52; Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), diluted 1/2000 in 0.1 M phosphate-buffer saline solution supplemented with 0.3% Triton X-100 and 0.25% bovine serum albumin. Sections were then incubated for 1 h with a biotinylated goat anti-rabbit immunoglobulin diluted 1/500, and for 1 h with an avidin-biotin complex (ABC, Novostain Super ABC Kit, Novocastra Laboratories, Newcastle, United Kingdom). Peroxidase activity was detected by using VECTOR NovaRED (Substrate Kit for Peroxidase, Vector Laboratories, Burlingame, CA, United States). Sections were mounted in sequential caudo-rostral order on slides, air-dried, dehydrated with absolute alcohol, cleared with xylene and coverslipped with mounting medium (Entellan^®, VWR International S.A.S).

Deparaffinized sections of livers (3 μm thick) were hydrated and heat epitope retrieval was performed in microwave oven in retrieval solution buffer pH = 6. After cooling to room temperature (RT), the slides were incubated with 0.3% solution of H₂O₂, washed twice with PBS and further incubated with 2.5% horse serum. After washing in PBS slides were incubated with primary antibody: rabbit anti-mouse Ki67 (Novus, USA) and rabbit anti-mouse Bax (Santa Cruz Biotech., USA) for 1 h in RT. After washing in PBS immunoreaction was visualized with ImmPRESS UNIVERSAL REAGENT and Vector NovaRED Substrate KIT FOR PEROXIDASE (VECTOR LABORATORIES, USA) according to manufacturer protocol. As a negative control, the primary antibody was replaced with PBS on the specimen. Positive staining was defined by visual identification of a yellow/brown pigmentation in the light microscope. Images were collected with an Olympus IX81 inverted microscope (Olympus) with color camera and with CellSens image processing software (Olympus).

Immunohistochemical analysis of vaspin was carried out by rehydrating deparaffinized uterine scrapings (3 μm thick) and performing thermal epitope recovery in a microwave oven in solution recovery buffer pH = 6 (DAKO Agilent, Santa Clara, CA, USA). After cooling to room temperature (RT), the slides were incubated with 0.3% oxidized water solution, then washed twice with PBS (phosphate-buffered saline) and further incubated with 2.5% horse serum (Vector Laboratories Newark, CA, USA). After incubation with serum, the slides were incubated with rabbit primary antibodies against human vaspin for one hour at RT. After washing in PBS (EURx, Gdańsk, Poland), immune reactions were visualized using ImmPRESS UNIVERSAL REAGENT and Vector Nova RED Substrate KIT FOR PEROXIDASE (Vector Laboratories Newark, CA, USA) according to the protocol posted by the manufacturer. The primary PBS antibody was substituted as a negative control in the sample. Positive staining was determined by visual identification of yellow-brown pigmentation by light microscopy. Images were collected using an Olympus IX81 inverted microscope (Olympus, Germany) with a color camera and CellSens image processing software 1.16 (Olympus, Germany).