Ab416

After behavioral tests, rats were decapitated. PFCs were quickly separated, cut into pieces, digested by papain with 0.1% DNase. Cell debris was removed and all cells were enriched by Percoll (17-0891-02, GE Healthcare). The isolated cells were fixed by 1% PFA. The fixed cells were incubated with primary antibodies rabbit anti-GLAST1 (1:100, ab416, Abcam) and mouse anti-GLUT1 (1:100, ab40084, Abcam) in permeabilized (0.1% Triton, for total GLUT1 detection) or non-permeabilized (without Triton, for plasma membrane GLUT1 detection) condition for 1 h. Then, cells were incubated with secondary antibody Alexa Flour 488 goat anti-rabbit IgG and Alexa Flour 647 goat anti-mouse IgG for 0.5 h. The fluorescence signal was detected by flow cytometer (Attune NxT, Invitrogen). Astrocytes were identified by GLAST1 expression (Norden et al., 2016 (link)).

Histological examination, whole-mount and section immunostaining and ISH were carried out according to standard procedures. Briefly, inner ears were fixed in 4% paraformaldehyde (PFA) for 1 hr at 4°C, dehydrated, and embedded in wax. Paraffin sections were generated at 6 μm. For ISH, tissues were fixed overnight. We used five embryos for each genotype at each stage for each probe and the result was consistent in each embryo.
Primary antibodies: anti-Sox2 (PA1-094, Thermo Fisher), -Myo7A (25–6790, Proteus and 138-1-s, DSHB), -Six1 (HPA001893, Sigma), -Atoh1 (Math1-s, DSHB), -p27^kip1 (554069, BD Pharmingen), -Calretinin (MA5-14540, Thermo Fisher), -p75^NTR (#07–476, EMD Millipore), -N-cadherin (610921, BD Bioscience), -E-cadherin (U3254, Sigma), -S100A (ab11428, Abcam), -GLAST (ab416, Abcam), -Pou4f3 (sc-81980, Santa Cruz), -Prox1 (AB5475, Millipore), -Acetylated tubulin (T7451, Sigma), -Cy3-, Cy2-, Cy5- and FITC-conjugated secondary antibodies were used. Alexa Fluor 488 or 350-conjugated phalloidin (A12379 and A22281, Life technologies) were used for actin staining. Hoechst 3342 was used for nuclear staining.

Hippocampal slices were washed 3 times with 0.01 M phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). At the end of fixation, the slices were washed 3 times with PBS and then stored at 4 °C in PBS containing 0.05% sodium azide until use. Prior to immunolabelling, slices were washed 3 times with PBS for 10 min, then incubated overnight with 1% Triton X-100 in PBS at 4 °C, followed by a blocking stage using 20% bovine serum albumin (BSA) diluted in PBS (0.01 M) and containing 0.1% Triton (PBS-T) for 3 h at RT. Slices were then incubated overnight with primary antibody diluted in PBS-T and 1% normal goat serum (NGS): rabbit anti-NeuN antibody (1: 200, Cell Signalling Technology, D4G4O # 24,307), guinea pig anti-GLT1 antibody (1:5000, Merck Millipore Chemicon International, ab1783), rabbit anti-GLAST antibody (1: 150, Abcam, ab416), or rabbit anti-GS (1:5000, Abcam, ab49873). Following washes in PBS-T, slices were incubated in appropriate secondary antibodies for 3 h at RT, namely goat anti-guinea pig Alexa-fluor 568 or goat anti-rabbit Alexa-Fluor 647 (both at 1:500, Invitrogen), in addition to the nuclear chromatin dye Hoechst 33,342 (1:500, Fisher, 11,544,876). Lastly, slices were washed in PBS-T (0.1%) and mounted in Fluoromount G (Invitrogen—REF 00–4958-02).