Animal free blocker and diluent

Immunohistochemistry (IHC) was performed to identify neuronal nuclei using Anti-NeuN (ABN90, EMD Millipore) and FITC anti-guinea pig (706–095-148, Jackson ImmunoResearch Laboratories Inc.) (Figure 6C). Primary and secondary antibodies were diluted to 1:500 with Animal-free Blocker and Diluent, R.T.U. (SP-5035, Vector Laboratories Inc.). Slides were kept at room temperature (RT) for 1 h, then washed for three 5 min cycles in PBS with gentle rocking. Next, slides were incubated in R.T.U. buffer for 12 h at 4°C. Following removal of excess buffer, the diluted primary antibodies were distributed between slides and incubated at 4°C for 36 h. Following three 10 min PBS wash cycles, slides were stained with secondary antibodies, diluted and distributed same as before, then incubated at 4°C for 24 h. After three 10 min PBS washes excess liquid was aspirated and glass coverslips were mounted using VectorShield medium with DAPI (H-1200, Vector Labs). Slides were kept in the dark at RT for 24 h before imaging.

After embedding in paraffin, sections of the otic bullae Sects. (4 µm) were prepared. They were subsequently deparaffinized and rehydrated. Antigen retrieval was performed in citraconic acid buffer (Immunosaver®, #097–06,192, Fujifilm Wako Pure Chemical Corporation) for 45 min in an autoclave at 95℃. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide for 15 min, and sections were blocked with an R.T.U. Animal Free Blocker and Diluent® (SP-5035, Vector Laboratories, Burlingame, CA, USA). After rinsing with phosphate buffer saline (PBS), the sections were incubated with rabbit polyclonal antibodies against ERα (1:200, #106,132-T08, Sino Biological) and 4-HNE (1:400, #bs-6313R, Bioss) overnight at 4℃. After subsequent rinsing with PBS once again, they were incubated with a secondary antibody (Histofine Simple Stain Mouse MAX-PO(R) #414,341, Nichirei Biosciences, Tokyo, Japan) for 1 h at room temperature and stained with the DAB chromogen for 10 min. After a final rinsing, counterstaining was performed using Mayer’s hematoxylin.

CB₁ and CB₂ receptor expression in the aorta wall was studied immunohistochemically followed by confocal microscopy. Cross sections (2 mm) of the aorta were fixed in 4% paraformaldehyde, embedded in paraffin, cut, place on a slide, deparaffinized, and rehydrated. Next, aorta sections were blocked (Animal-free blocker and diluent, Vector laboratories Inc., Burlingame, CA, USA) and incubated with rabbit polyclonal Anti-CB₁ (1:100, Alomone, Labs, Jerusalem, Israel) or rabbit polyclonal Anti-CB₂ (1:100, Alomone, Labs, Jerusalem, Israel) primary antibody and co-labeled with mouse monoclonal Anti-alpha smooth muscle Actin antibody (1:200, Abcam, Cambrige, UK) overnight. The next day, primary antibodies were removed, and the samples were incubated with secondary antibodies for 2 h (FITC anti-rabbit, 1:100, Abcam (Cambrige, UK); and Alexa Fluor-568 anti-mouse, 1:50, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were counterstained with DAPI. Then a coverslip was placed using an antifade reagent (Prolong Diamond Antifade, Invitrogen, Waltham, MA, USA). Finally, images were acquired using a confocal microscope (Mod LSM 700, Zeiss), 3-D blind deconvolved (AutoQuant X3, Media Cybernetics), and subsequently analyzed with ZEN2009 (Zeiss), and Image J software (National Institutes of Health, Bethesda, ML, USA).