Hrp conjugated anti rabbit igg

Femoral bones were decalcified with 10% EDTA-2Na and dehydrated in ascending ethanol solutions prior to paraffin embedding and sectioning. Dewaxed paraffin sections were examined for EBAG9 and TM9SF1. In brief, after inhibition of endogenous peroxidases with methanol containing 0.3% hydrogen peroxidase for 30 min, dewaxed paraffin sections were pretreated with 1% bovine serum albumin (BSA; Serologicals Proteins Inc., Kankakee, IL, USA) in PBS (1% BSA-PBS) for 30 min. Sections were then incubated for 2-3 h at room temperature (RT) with mouse monoclonal EBAG9 antibody diluted at 1:100 with 1% BSA-PBS or rabbit polyclonal TM9SF1 antibody (APR44683, AVIVA SYSTEMS BIOLOGY, San Diego, CA, USA) diluted at 1:100. They were followed by incubation with horseradish (HRP)-conjugated anti-mouse IgG (61-6520, Chemicon International Inc., Temecula, CA, USA) or HRP-conjugated anti-rabbit IgG (P0399, DakoCytomation, Glostrup, Denmark). For visualization of all HRP-conjugated immunoreactions, diaminobenzidine tetrahydrochloride (DAB) (Dojin chemical) was employed as a substrate.

The fixed tissues were embedded in paraffin and sectioned into 3 μm-thick sections. To evaluate spongiform changes, the sections were stained with hematoxylin (WAKO, 131-09665) and eosin (WAKO, 056-06722). For Iba-1 staining, after deparaffinisation and rehydration, the sections were boiled in Target Retrieval Solution, Citrate pH 6 (DAKO, S2369) for 20 min for antigen retrieval. The blocked sections were treated with 0.3% hydrogen peroxidase (WAKO, 086-07445) in methanol (Hayashi Pure Chemical, 130-02069) for 30 min to inactivate endogenous peroxidase and then incubated with 3% nonfat dry milk (Megmilk Snow Brand, FA-08) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature. The blocked sections were incubated with a primary antibody (anti-Iba-1 antibody, WAKO, 019-19741) overnight at room temperature, followed by envision polymer horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (DAKO, K4002) for 60 min at room temperature. Immunostaining was visualised using 3, 3-diaminobenzidine (DAB; Dojindo Lab, D006). The hydrolytic autoclaving and formic acid method for PrP^Sc staining have been previously described (Ishibashi et al., 2012 (link)).