Mouse immunohistochemistry and immunofluorescence

The mouse olfactory epithelium, the olfactory bulbs, and the brain were isolated and left in 30% sucrose until sunk to the bottom of the tube. After embedding in Tissue Tek OCT, the tissues were sectioned with a thickness of 14 μm. For the multiplex fluorescence labeling of NRP1 mRNA (RNAScope Probe-Mm-Nrp1, Advanced Cell Diagnostics, 471621), the RNAscope® Multiplex Fluorescent v2 Assay (Advanced Cell Diagnostics, 323100) was used according to the manufacturer’s instructions. For the immunohistochemistry, the sections were air dried overnight at room temperature, washed in PBS and permeabilized with 0.1% Triton-X-100 for 15 min at room temperature. The blocking performed with 1% fetal calf serum, 1% fish gelatin and 1% bovine serum albumin in PBS for one hour at room temperature, followed by a second blocking step for endogenous mouse Ig with AffiniPure Fab fragment Donkey anti-
mouse IgG (Biozol, JIM-715-007-003) diluted 1:100 in blocking solution for one hour at room temperature. Primary antibodies were diluted in 10% blocking solution: 1:250 NRP1 (monoclonal rabbit, ab81321, Abcam); 1:1000 TuJ1 (monoclonal mouse, G712A, Promega); 1:250 NeuN (polyclonal chicken, ABN91, Milipore); 1:2000 GFP (polyclonal rabbit, A-6455, Thermo Fisher Scientific), 1:250 ColIV (polyclonal goat, 1340-01, Southern Biotech), 1:250 CD31 (monoclonal mouse, sc-376764, Santa Cruz Biotechnology), 1:500 AQP4 (polyclonal rabbit, AQP-004, TMH-Medizinhandel) and incubated overnight at 4°C. After three washes in PBS, the sections were incubated in secondary antibody: Alexa Fluor 488 donkey anti-mouse (R37114, Thermo Fischer Scientific); Alexa Fluor 488 goat anti-rabbit (A21428, Thermo Fischer Scientific); Alexa Fluor 488 goat anti-chicken (A11039, Thermo Fischer Scientific); Alexa Fluor
488 donkey anti-goat (A11055, Thermo Fischer Scientific); Alexa Flour 555 donkey anti-goat (A21432, Thermo Fischer Scientific); Alexa Flour 555 goat anti-mouse (A21422, Thermo Fischer Scientific); Alexa Flour 647 donkey anti-rabbit (A31573, Thermo Fischer Scientific), Alexa Fluor 488 Phalloidin (A12379 Thermo Fischer Scientific), for two hours at room temperature. After three washes in PBS, the sections were counterstained with Hoechst 33342 stain solution (Thermo Fisher Scientific) and mounted with Prolong Gold Antifade Mountant (Thermofisher, P36930). For immunofluorescence, the cells were permeabilized on ice for 10 minutes in 0.1% Triton-X-100 and 0.1% Sodium citrate in PBS, blocked for one hour in 1% fetal calf serum, 1% fish gelatin and 1% bovine serum albumin in PBS at room temperature and left overnight at 4°C in primary antibody diluted in 10% blocking solution. After three washes in
PBS, the cells were incubated with secondary antibodies (diluted 1:500 in 10% blocking solution) for one hour at room temperature. After three washes in PBS, the cells were stained with Hoechst 33342 (Thermo Fisher Scientific) and mounted with ProLong Gold Antifade. The stained tissues and cells were imaged on a Leica SP5 confocal microscope.