Alexa fluor 594 and 488 conjugated secondary antibodies

Frozen muscle cryosections were stained using rabbit anti mouse-MyoD (M-318, SantaCruz) and monoclonal mouse anti-myogenin (F5D, DSHB) antibodies (green) were visualized with Alexa Fluor-594- and -488-conjugated secondary antibodies (Invitrogen) before mounting in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). Macrophages sorted from injured muscle on day 4 post injury through MACS technology were spun onto glass slides and kept at −80 °C until use. Apolipoprotein E immunolabelling used a rabbit polyclonal anti-mouse ApoE (Abcam, 1/30^e), and an anti-rabbit Alexa 488 antibody (Life Technology) as a secondary antibody.

Cells seeded on coverslips in 12-well plates were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. After that, the cells were incubated with anti-UBE2M and anti-β-catenin at 4 °C for overnight. Subsequently, Alexa Fluor 594- and 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were incubated with the cells at 37 °C for one h. The cells were stained with 4',6-diamidino-2-phenylindole (DAPI), and photographed using a Leica SP8 confocal microscope (Leica, Germany).

Antibodies used were as follows: anti-118Y-p-paxillin, anti-31Y-p-paxillin and anti-397Y-p-FAK (Invitrogen); anti-GFP, anti-β-actin, anti-Rab7, anti-p62 and anti-LC3B (Santa Cruz); anti-416Y-p-Src and anti-NBR1 (Cell signaling), anti-GFP (Roche); anti-LAMP-1 and anti-HA (Abcam); anti-LC3B, anti-Atg12 and anti-c-Cbl (Novus); anti-Rab5a (BD transduction laboratory); anti-paxillin, anti-Src and anti-FAK (Millipore); anti-LAMP1 (R&D systems); anti-FLAG (Sigma). Anti-mouse and anti-rabbit IgG-peroxidase-conjugated secondary antibodies for Western blot assays were from Bio-Rad. Alexa Fluor594 and 488 conjugated secondary antibodies were from Life Technology. Alexa Fluor 647 conjugated secondary antibodies were from Millipore. Epidermal growth factor was from Gibco; Histodenz (Nycodenz), nocodazole and chloroquine (CQ) were from Sigma-Aldrich. When indicated CQ was used at a concentration of 20 μM, a non-toxic concentration found to induce optimal autophagy inhibition (based on changes in LC3I/II ratio), which is in accordance with previous studies (Sandilands et. al.).

Following an overdose of pentobarbital, rats or mice were transcardially perfused using PBS followed by 4% PFA in PBS. Brains were post-fixed during 24 h in 4% PFA, followed by a dehydration ethanol series and paraffin embedment. Coronal sections (8 µm) were cut at the level of the hippocampus (−1.8 mm from bregma) in mice and rats, and at the level of the lateral ventricles (bregma) in rats. These brain areas were selected as we observed deficits in the white matter here previously in the respective models [32 (link),50 (link)]. For immunofluorescence stainings, sections were deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. For antigen retrieval, sections were heated to 95 °C in sodium citrate buffer (0.01M, pH 6). After cooling down and three PBS/0.05% Tween20 (PBS-T) washes, non-specific binding was blocked using 5% normal donkey serum in PBS-T. Subsequently, sections were incubated overnight with primary antibodies diluted in PBS (see Table 2 for details and dilutions). The next day, sections were washed three times in PBS and incubated with alexa fluor 594 and 488 conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA; 1:500) for 75 min at room temperature. Sections were counterstained using DAPI (1:5000) and embedded in FluorSave (Merck Millipore, 345789, Burlington, MA, USA).