Novocastra dab chromogen

RNA scope on fresh murine sections was performed using RNAscope 2.5 HD Detection Reagent-BROWN (Advanced Cell Diagnostic) in accordance with the manufacturer’s protocol. Mouse Prdm1 (#441,871) was used as probe. Novocastra DAB Chromogen (Leica Biosystems, #RE7105) in its Novocastra DAB Substrate Buffer (Leica Biosystems, #RE7106) was used to reveal signals.

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.

Immunohistochemistry was performed using the peroxidase reaction technique with identification by polymerized secondary antibody (Novocastra Post Primary and Novolink Polymer; Leica Biosystems, UK). The antigen retrieval was performed by pressurized humid heat at 137°C (Autoclave ALT 5LD Plus; ALT, Brazil) with Target Retrieval Solution Citrate - low pH (Agilent Technologies, USA). Blockage of endogenous peroxidase and endogenous proteins was done with Novocastra reagents (Leica Biosystems). Antibodies anti-Ca_V3.1, -Ca_V3.2, and -Ca_V3.3 (ACC-021, ACC-025, and ACC-009; Polyclonal, Alomone Labs, Israel) were used in dilutions of 1:250, 1:100 and 1:200, respectively. Incubation with primary antibody was set at 16 h and with 3'3-diaminobenzidine chromogen Novocastra DAB Chromogen (Leica Biosystems) at 1 min. Sections were counterstained with hematoxylin dye. For positive controls of Ca_V 3.1 and Ca_V3.2 labelling, we used samples of human skeletal muscle, and for Ca_V3.3, samples of human brain were used. For negative controls, the primary antibody incubation step was replaced by incubation with immunoglobulins of the same species as the primary antibody.