Alexa fluor 647

For high-resolution imaging of IHC ribbons, we used a Leica DMI6000 TCS SP8 STED microscope with 93X glycerol immersion objective (NA 1.3) (Bordeaux Imaging Center). Mouse sensory organs were fixed and incubated with primary anti CtBP2 (1/100) antibodies in conditions similar to those described above for confocal microscopy. Secondary antibodies Goat anti- IgG1 Mouse polyclonal Alexa Fluor 647 (Jackson, cat# 115-605-205) were used to label CtBP2 primary antibodies. The pulsed STED depletion laser (pulsed laser IR, Spectra Physics Mai Tai) was set at 775 nm. Stack images were acquired with the following parameters: scan rate 200 Hz, 775 nm depletion laser power between 8 and 15%, scans frames accumulated per X–Y section 8 times, z-step size 0.25 μm, number of plans between 8 and 10, pixel size 20 nm giving an X–Y image size of 21 × 5 μm (1,024 × 256 pixels).

Cells on coverslips were fixed in a 1:1 mixture of methanol and acetone at -20 °C for 5 minutes and blocked in 10% normal goat serum and 1% BSA in PBS for 20 minutes. Primary antibodies (mouse monoclonal RR90, recognising FLNc and FLNa58 ; mouse monoclonal XR1, recognising XinA and XinB59 (link); rabbit serum RaA653 against sarcomeric α-actinin58 ) diluted in PBS containing 0.05% Tween (PBST) were applied on the coverslips for 1 hr in a moist chamber. Coverslips were washed twice by immersion in PBST, incubated with secondary antibodies conjugated to FITC, Cy3 or Alexa Fluor 647 (Jackson ImmunoResearch/Dianova, Hamburg, Germany) for 30 minutes and washed as before in PBST and once in distilled water before mounting in Mowiol containing 10% N-propyl gallate. Tissue cryosections of soleus muscles from exercised mice on slides were fixed with methanol (2 min) followed by acetone (20 sec) at −20 °C and blocked as above for 30 minutes. Primary antibodies (as above) were applied in 1% BSA in PBS consecutively overnight at 4 °C, slides were washed twice in PBST and once in PBS (5 minutes each) and secondary antibodies (as above) were applied in 1% BSA in PBS consecutively at 37 °C for 2.5 hrs each. Slides were washed as before and gently rinsed in distilled water before mounting as above.

The slides were incubated overnight at 4° C with primary antibodies for Par2 (1/200, mouse, Thermos Fisher Scientific, San Diego, CA, USA), CD45 (1/200, rat, Novus Biological, Centennial, CO 80112, USA), and albumin (1/100, gout, R&D Systems Inc., Minneapolis, MN, USA). Secondary antibodies were labeled with Alexa Fluor 488, Alexa Fluor 647, and Rhodamine Red (all from Jackson ImmunoResearch). Nuclei were visualized with DAPI Fluoromount-G^TM (Bar Naor Ltd, Ramat Gan, Israel). The slides were imaged using Microscope Axio Imager M2 (Zeiss).

Rabbit anti-Synaptotagmin Dsyt CL1 (α-SYT; 1:5000; Dr Noreen Reist, Colorado State University, Fort Collins, CO; Mackler et al., 2002 (link)); rabbit anti-dEAAT1 (1:2500; Dr Serge Birman, Developmental Biology Institute of Marseille, France; Rival et al., 2006 (link)); monoclonal antibody (mAb) nc82 anti-BRP (BRUCHPILOT; 1:50; Developmental Studies Hybridoma Bank, Iowa City, IA; Wagh et al., 2006 (link)); mAb GS-6 anti-Glutamine Synthetase (α-GS2) (MAB 302; 1:500; EMD Millipore, Billerica, MA); goat anti-HRP antibody conjugated to Alexa-Fluor-647, which labels neuronal plasma membranes (123-605-021; 1:200; Jackson Immunoresearch Laboratories, West Grove, PA); mAb FK2 anti-Ubiquitin (BML-PW8810-0100; 1:1000; Enzo Life Sciences, Farmingdale, NY); rabbit anti-P62 (1:5000; Dr Gábor Juhász, Eötvös Loránd University, Hungary; Pircs et al., 2012 (link)); mAb 6-11B-1 anti-Tubulin (T6793; 1:20,000; Sigma-Aldrich, St. Louis, MO); mAb YL1/2 anti-Tubulin (ab6160; 1:500; Abcam, Cambridge, UK) and rabbit anti-SERCA (1:200; Subhabrata Sanyal, Biogen, Cambridge, MA; Sanyal et al., 2005 (link)) antibodies. Actin staining was performed using Alexa-Fluor-568-conjugated phalloidin (A12380; 130 nM; Invitrogen, Carlsbad, CA), and nuclear staining was performed using DAPI (D9542; 300 nM; Sigma-Aldrich, St. Louis, MO).