Super frost plus cryoslides

The head was fixed for 2 h at 4°C in fixative solution, cryoprotected in 30% sucrose, and embedded in Leica tissue freezing medium (Leica Microsystems). Sections of 12 μm thickness were cut on a Leica CM 1900 UV cryostat (Leica Microsystems), adhered to Super Frost® Plus cryoslides (Thermo Scientific) and stored at −25°C.

Tissue was fixed in 4% PFA solution overnight at 4 °C, transferred to 30% sucrose solution for cryopotection, frozen, and embedded in Tissue Freezing Medium (Leica Biosystems, 14,020,108,926). Coronal sections of 16-µm thickness were made using a Leica CM3050S Cryostat (Leica Biosystems, Wetzlar, GER) and collected onto SuperFrost Plus™ cryoslides (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER). Antigen retrieval was performed with citrate buffer (0.1 mol/l Tri-sodiumcitrate dehydrate, 0.5% Tween 20, pH 6) at 97–99 °C in a steamer (Tefal Vitacuisine, Sarcelles, FRA). The sections were incubated in blocking buffer (2% BSA, 3% Normal Donkey Serum, 0.1% Triton in TBS) with antibody solutions (Additional file 1: Table S1) and mounted with coverslips using Fluoromount-G™. For en face labelling, the tissue was fixed in 4% PFA for 15 min, the OE was separated from the septum by gently lifting it off with a pair of fine tweezers and transferred onto a Polysine adhesion slide (Thermo Fisher Scientific Germany Ltd. & Co. KG, Bonn, GER; J2800AMNZ) such that the ciliary side was on top. X-Gal Staining was performed with a kit (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, GER) according to the manufacturer’s instructions.