Rabbit anti laminin

Immunocytochemistry was performed as previously described (Spohr et al., 2011 (link)). Briefly, cultured cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 5 min at 24°C. After permeabilization, the cells were blocked with 5% bovine serum albumin (BSA) (Sigma Chemical Co.) in PBS (blocking solution) for 1 h and incubated overnight at 4°C with specified primary antibodies diluted in blocking solution. Primary antibodies were mouse anti-β-tubulin III antibody (Promega Corporation, Madison, WI, USA; 1:1000), rabbit anti-fibronectin (Sigma Chemical Co., 1:100), and rabbit anti-laminin (Sigma Chemical Co., 1:100). After primary antibody incubation, cells were extensively washed with PBS and incubated with secondary antibodies for 1 h, at 24°C. The secondary antibodies were goat anti-mouse IgG conjugated with alexa fluor 488 (Molecular Probes, Eugene, OR, USA; 1:500) and goat anti-rabbit IgG conjugated with alexa fluor 546 (Molecular Probes, 1:500). Negative controls were performed by omitting the primary antibody during staining. In all cases, no reactivity was observed when the primary antibody was absent. Cell preparations were mounted directly on n-propyl gallate (Sigma Chemical Co.) and visualized using a Nikon microscope.

OBSCs were fixed for 20 min in 4% PFA in phosphate buffered saline (PBS), washed three times in PBS, then blocked for 1 h in PBS supplemented with 0.5% Triton X-100 and 3% normal Goat Serum (Sigma G9023). Slices were incubated at 4 °C in primary antibody (diluted in blocking solution) overnight. In order to detect PDGFRβ, heat-mediated antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 40 min at 80 °C prior to primary antibody incubation. Slices were washed a further three times in 0.5% Triton-X100 in PBS (PBS-TX) then incubated with secondary antibodies (Life Technologies and Jackson) (1:500 dilution in blocking solution for 2 h at 4 °C). Three final PBS-TX washes were conducted before slices were mounted on slides and images captured using a Leica Confocal Microscope. Primary antibodies used: rabbit anti-PDGFRβ (28E1) (1:200, Cell Signalling, Cat. No: 3169S), rat anti-PECAM-1 (1:400, BD, Cat. No: 550274), rabbit anti-laminin (1:200, Sigma, Cat. No: L9393), Mouse MOAB-2 (pan Aβ) (1:1000, Merck-Millipore, Cat. No: MABN254) Rabbit Ki67 (1:1000, Abcam, Cat. No: ab15580) secondary staining was conducted using species-specific fluorophore-conjugated (Streptavidin Alexa 488, Molecular Probes; Cy3 or Cy5, Jackson,) or biotin-conjugated secondary antibodies (Jackson). DAPI (1 μg/mL, Sigma) was used to counterstain nuclei.

Formalin-fixed muscles were bisected and paraffin-embedded, and 10-μm cross sections were taken at the midbelly region. Sections were deparaffinized, rehydrated and subjected to Masson’s trichrome stain. In regard to immunohistochemitry, deparaffinized and rehydrated10-μm cross sections were subjected to antigen retrieval by heating in Rodent Decloaker solution (BioCare Medical, Pacheco, CA) at 80 °C for 30 min. Sections were blocked in 5% goat serum in PBS and incubated in rabbit anti-laminin (Sigma) diluted in 1:100 in blocking solution. Sections were then incubated in goat anti-rabbit secondary antibody conjugated to Alexa 488 (Life Technologies) diluted 1:200 in blocking solution. For detection of type II fibers sections were further incubated in anti-fast (type II) MHC conjugated to alkaline phosphatase (Sigma, St. Louis, MO) diluted 1:50 in PBS for 60 min at room temperature. Whole stained sections were scanned on an Olympus VS120 fluorescent slide scanner.

Embryos were fixed in 4% PFA, rinsed in PBT, digested briefly in 10 μg/mL proteinase K, refixed in 4% PFA, rinsed in PBT, and blocked in 2 mg/mL BSA + 2% goat serum in PBT. Embryos were then incubated overnight in rabbit anti-Laminin (Sigma L9393) at 1:200, mouse anti-Myc (Cell Signaling 2276) at 1:1000, or rabbit anti-phospho histone H3 (Upstate 06-570) at 1:500 in blocking solution, rinsed in PBT, and incubated overnight in Alexa Fluor 488 anti-Rabbit IgG, 568 anti-Rabbit, or 568 anti-Mouse (Invitrogen) at 1:1000 in PBT. Embryos were co-stained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride and rinsed in PBT prior to mounting in agarose for confocal imaging.

Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).

Muscle sections, single myofibers, and cultured cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in 0.3% Triton X-100 in PBS. The samples were blocked in blocking buffer (Beyotime Biotechnology) for 2 h at room temperature. Primary antibodies were incubated in blocking buffer at 4 °C overnight. Subsequently, the samples were washed with PBS and stained with the appropriate fluorescently labeled secondary antibodies (Alexa Fluor 350, 488, or 594) for 1 h at room temperature. After washing with PBS, DAPI (Roche) was used to stain nuclei for 3 min. The primary antibodies used were as follows: mouse anti-Pax7 (Developmental Studies Hybridoma Bank [DSHB], 1:50), mouse anti-eMyHC (DSHB, 1:50), mouse anti-MyoG (cat. no. ab1835, Abcam, 1:200), mouse anti-MyHC (cat. no. M4276, Sigma, 1:500), rabbit anti-laminin (cat. no. L9393, Sigma, 1:200), rabbit anti-Dvl2 (cat. no. ab22616, Abcam, 1:200), rabbit anti-β-tubulin (cat. no. ab6046, Abcam, 1:500), mouse anti-Flag (cat. no. F1804, Sigma, 1:500), and rabbit anti-HA (cat. no. ab9110, Abcam, 1:200). For live-cell staining, DiI (5 μM, Beyotime Biotechnology) and Hoechst 33342 (2 μg/ml, Beyotime Biotechnology) were applied for 20 min.