Cy3 conjugated goat anti rabbit igg

To evaluate the cellular distribution of pre-miR-7-2 in the brain, FISH assay was performed based as our previous description with some modifications [23 (link)]. Briefly, before hybridization incubation, all solutions were prepared with diethylpyrocarbonate-treated water. After deparaffinization and rehydration, tissue sections were treated by pepsin digestion. Sections were next incubated or heated in the microwave, and then were incubated with hybridization cocktail containing miR-7-2 probe (1:1000; EXIQON; no. 38485–01) at 42 °C for overnight. Next, the sections were washed in PBS and incubated with a secondary antibody of Cy3 conjugated goat-anti-rabbit IgG (1:250; Invitrogen) in the dark, at room temperature for 1 h. Then, the slides were rinsed with PBS-T three times, for 5 min each and counterstained, mounted with Slow Fade Gold Antifade Reagent with DAPI (1:1000) in the dark, at room temperature for 10 min, before examination by fluorescence microscopy (Zeiss Axioplan 2).

IF and IHC were carried out on paraffin-embedded samples after dewaxing and rehydration. For IF staining, the lung sections were incubated with primary antibody against NOD-like receptor family pyrin domain containing 3 (NLRP3) (ABclonal Biotechnology, Wuhan, China) at a dilution of 1 : 100 at 4°C overnight and secondary antibody against Cy3-conjugated goat-anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) at a dilution of 1 : 200 at room temperature for an hour. 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin regents Co. Ltd., Shanghai, China) was used for for nuclear labeling. The sections were examined under a microscope.
The sections for IHC staining were treated with 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 15 min and blocked by 1% bovine serum albumin (BSA) for 15 min. Then, sections were immunostained with primary antibody (TGF-β1, Affbiotech, Changzhou, China) and secondary antibody (HRP-conjugated goat-anti-rabbit IgG, Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). Diaminobenzidine (DAB) (Maxim Biotech, Fuzhou, China) was employed as a developer, and hematoxylin was used for counterstaining. IHC images were captured under a microscope.