Envision system hrp labelled polymer anti mouse

Samples were fixed in formalin and embedded in paraffin before being sectioned and immunostained. Three μm thick slides were incubated at 60 °C for an hour prior to staining. Before staining, the sample antigen retrieval was done in a buffer with either citric buffer (pH 6) or TE buffer (pH 9) for 20 min. Samples were then blocked with serum. The primary antibody was incubated overnight at 4 °C and the secondary antibody incubated at room temperature for 30 min. Secondary antibodies used included Dako EnVision+ system-HRP labelled polymer anti-mouse (K400011–2), anti-rabbit (K400211–2) and the DAB substrate kit (ab94665). A kit containing DAB chromogen and substrate buffer (ab94665) was used according to the manufacturer’s instructions.

Human investigations were performed with informed consent and were preceded by local institutional review board approval.
Samples were routinely fixed in 10% formaldehyde buffer (pH 7.4) for 24 hrs, paraffin-embedded, and processed for immunohistochemical analysis. Slides were incubated with anti-p130Cas (Labvision Thermo Scientific) (1 microg/mL) for 1 hr at room temperature, after antigen retrieval (citrate buffer, at 98°C for 40 min). Staining was detected with EnVision System-HRP Labelled Polymer anti-mouse (DakoCytomation) and developed with the LiquidDAB Substrate Pack (BioGenex, San Ramon, CA, USA). Nuclei were counterstained with Mayer hemallum. Images were taken using a Leica DM 2000 microscope.

Paraffin-embedded sections were used for histologic and IHC staining. Changes in renal morphology were examined by H&E staining. Matrix deposition within the interstitium was assessed using Masson’s trichrome staining. For IHC staining, kidney slides were incubated in citrate buffer (pH 6, heated to 99°C) for epitope retrieval and 0.3% hydrogen peroxide to block endogenous peroxidase activity. After preincubation with 10% protein block (Dako) to block nonspecific binding of antibodies, the tissues were incubated overnight at 4°C with primary antibodies against COL-1 (Abcam, 34710), α-SMA (MilliporeSigma, A2547), FN (Abcam, 45688), COL-4 (Abcam, 6586), COL-3 (Abcam, 7778), CXCR4 (Santa Cruz Biotechnology, sc-53534), TGF-β (Santa Cruz Biotechnology, sc-130348), and LOXL2 (Santa Cruz Biotechnology, sc-66950). Slides were then washed and incubated with secondary antibodies EnVision+System-HRP Labelled Polymer Anti-rabbit (Dako, K4003) and EnVision+System-HRP Labelled Polymer Anti-mouse (Dako, K4001). After washing with TBS-Tween 20, kidney sections were covered with DAB (Dako) for 10 minutes to produce a brown color. Ten randomly chosen fields of kidney cortex were captured per mouse, and staining was quantified as percentage of total area, using ImageJ.