Novolink dab substrate buffer

Formalin-fixed paraffin-embedded sections were dewaxed in xylene and ethanol, and subsequently washed with dH₂O. Antigen retrieval was performed in a pressure cooker using TRIS-EDTA buffer (10 mM TRIS, 1 mM EDTA, 0,05% Tween20, pH = 9) for 30 min. After cooling slides were washed with PBS, and endogenous peroxidase was inactivated by addition of 10% H₂O₂ in methanol for 20 min. To prevent any nonspecific binding 5 w/v% bovine serum albumin (BSA) containing 10 v/v% normal serum dissolved in PBS was applied for 1 h at room temperature. Monoclonal Mouse anti-human CD138 Clone M15, (Dako Agilent, Santa Clara, CA) diluted at 1:50 in 1 w/v% BSA/PBS was applied overnight at 4 °C. The next day, slides were washed in PBS containing 0,05% Tween-20, then horse radish peroxidase conjugated Polyclonal Goat anti-Mouse Ig HRP (Dako Agilent) at 1:200 dilution was used for 1 h at room temperature. The immunoreaction was detected by Novolink DAB Substrate Buffer, according to the manufacturers recommendation (RE 7143, RE 7105, Leica Biosystem, Wetzlar Germany) [13 (link)].

IHC was used to localize the NRN1 protein qualitatively in eutopic endometrium and endometriotic tissues from women with endometriosis. IHC was performed using a Novolink Polymer Detection System (RE7140-K; Leica Biosystems, Newcastle Upon Tyne, UK). Paraffin blocks of each sample were cut at a thickness of 3 μm. Eutopic endometrial tissues from patients without endometriosis (normal endometrium) were used as control. Routine hematoxylin and eosin staining were performed to provide a tissue overview. Sections were deparaffinized and dehydrated, and antigen retrieval was performed in Novocastra Epitope Retrieval Solution, pH 6.0 (Leica Biosystems). The tissue sections were then processed using the kit according to the manufacturer’s protocol and were incubated with the primary antibody (rabbit polyclonal anti-NRN1 antibody; ab64186; dilution, 1:100; Abcam) overnight at 4 °C. To develop the peroxidase activity, sections were incubated with 3,3′-diaminobenzidine (DAB) working solution (Novocastra DAB Chromogen and Novolink DAB Substrate Buffer, Leica Biosystems) and counterstained with Novocastra Hematoxylin (Leica Biosystems). Negative controls were treated identically with the exception that the primary antibody was replaced with a rabbit IgG isotype control (ab199376; Abcam). Images were captured using the SPOT imaging software.