X gal

Cryosections from gastrocnemius muscles or mouse kidney were fixed with 1% paraformaldehyde/0.2% glutaraldehyde (Electron microscopy sciences). Slides were incubated with PBS, pH 6.0 for 30 minutes, followed by incubation with X-gal staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM magnesium chloride, 0.02% Igepal CA-630, and 400 μg/mL X-gal; all from Sigma) for 48 h at 37 °C. Slides were washed with PBS, and fixed in 1% paraformaldehyde. Slides were washed with PBS and coverslips were mounted with Fluromount G (SouthernBiotech). Slides were imaged on a Nikon-Ni widefield microscope using a Nikon DS-F12 color camera.

X-gal staining was performed on 9.5 dpc, 10.5 dpc, 11.5 dpc, 13.5 dpc Pet1₂₁₀-Cre/ROSA26R whole embryos and on P1 or P10, P30 and adult Pet1₂₁₀-Cre/ROSA26R kidney and brains, respectively. Dissected tissues or whole embryos were fixed in 2% formaldehyde solution prepared in PBS for 30 minutes, and subsequently processed for X-gal staining solution containing 5 mM K₄Fe(CN)₆, 5 mM K3Fe(CN)₆, 2 mM MgCl₂, 0.2% NP40, 0.1% sodium deoxycholate and 1 mg/ml X-gal (Sigma) in PBS for 4–16 h at 30°C. Samples were post-fixed in 4% PFA at 4°C o/n.
β-galactosidase stained specimens were cut at 50 µm with a vibratome or clarified in methyl salicylate pure solution to enhance contrast between X-gal staining and non-stained tissues.

Semi-thin tissue cryosections were washed in PBS for 10 min shaking and incubated for 10 min in permeabilizing solution (0.01% (w/v) Na-deoxycholat, 0.02% (w/v) NP-40 in PBS). After a washing step in PBS, the samples were incubated for 3 hr in 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal)- solution (5 mM K₃Fe(CN)₆, 5 mM K₃Fe(CN)₆, 2 mM MgCl₂ and 1 mg/ml X-Gal (Sigma-Aldrich)) at 37°C with gentle shaking. The sections were washed twice in PBS and embedded in mounting medium.

AP cells fixed with 4% paraformaldehyde (Thermo Fisher Scientific) were incubated with X-gal solution (40 mM citric acid/Na phosphate buffer (Sigma-Aldrich)), 5 mM K4[Fe(CN)6]0.3H2O (Sigma-Aldrich), 5 mM K3[Fe(CN)6] (Sigma-Aldrich), 150 mM sodium chloride (CIMR), 2 mM magnesium chloride (Sigma-Aldrich) and 1 mg/ml X-gal (Sigma-Aldrich) in distilled water) for 16 hours at 37°C. Cells were washed twice with 1X DPBS. Images were captured at 40X magnification using the Olympus DP20 microscope camera attached to an Olympus CKX41 light microscope.

For X-gal staining, brain slices and isolated cochleae, slit from the apex to base, were incubated with the β-galactosidase staining solution containing 0.5 mg/ml X-gal (Sigma) as described (Chumak et al., 2016 (link)). Whole-mount X-gal staining of mouse embryos was performed as described previously (Ter-Avetisyan et al., 2014 (link)). In brief, embryos were fixed, their tissues equilibrated and stained in β-gal wash solution containing 0.5 mg/ml X-gal and 5 mM potassium ferrocyanide and ferricyanide. After development of a blue color, the reaction was stopped and after washing in PBS, the probes were post-fixed in 4% paraformaldehyde (PFA) and further processed for clearing before microscopic analysis. For cochlear staining of adult mice, isolated cochleae, slit from apex to base, were incubated with the β-gal staining solution containing 0.5 mg/ml X-gal (Sigma), 5 mM K₃[Fe(CN)₆], and 5 mM K₄[Fe(CN)₆] in PBS complemented with 20 mM MgCl₂, 0.01% sodium deoxycholate, and 0.02% Nonidet-P40 overnight at 37°C. After incubation, the cochleae were decalcified, embedded, and cryosectioned as described (Zuccotti et al., 2012 (link)). The presence of β-gal protein was visualized with the enzyme’s substrate (X-gal), resulting in a blue precipitate only in cells which express the inserted lacZ reporter cassette in exon 1 of the GC-B gene.