Blocking reagent

Mouse pancreata were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained using conventional histological techniques. Tissue sections (5 μm) were deparaffinized and retrieved using the 2100-Retriever (Electron Microscopy Sciences, Hatfield, PA) with antigen unmasking solution (Vector Laboratories, Burlingame, CA). For IHC, sections were incubated in 3% H₂O₂ for 5 min to block endogenous peroxidase activity followed by 1 hr in M.O.M. blocking reagent (Vector Laboratories, Burlingame, CA). Tissue sections were incubated in primary antibodies for 1 hr at room temperature. Biotinylated secondary antibodies were used at 1:200 dilution for 20 min at room temperature. IHC development was performed using Vector reagents and DAB (diamonibenzidine) peroxidase substrate (Vector Labs, Burlingame, CA). Secondary antibodies for immunofluorescence utilized avidin-conjugated Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 555, Oregon Green 488, and Alexa Avidin Cy5.5 (Invitrogen, Camarillo, CA). Detailed information on the primary antibodies used in this study is provided in S2 Table

Mice were euthanized and TA muscles were harvested, weighed, and embedded in OCT, and frozen in liquid nitrogen-cooled isopentane. Embedded muscles were transversely sectioned at 10 µm thickness. Sections were post-fixed in 4% PFA/PBS 10 min at room temperature, permeabilized in 0.1 M glycine, 0.1% Triton X-100 in PBS, and blocked in 5% goat serum, 2% BSA in PBS supplemented with M.O.M. Blocking reagent (Vector Laboratories). Then, sections were incubated with primary antibodies as described in “Immunostaining on cells, myofibers, and sections”. Muscle cross-sections stained with anti-Laminin and anti-Dystrophin and counterstained with DAPI were analyzed for fiber counting and minimum Feret’s diameter using SMASH^{63 (link)}, and centronuclei per myofiber were quantified using MuscleJ^{64 (link)}.

Upon deparaffinization and hydration, the slides underwent heat-induced antigen retrieval using citrate buffer-based antigen unmasking solution (Abacus) at 100 °C for 10 min. Mouse on Mouse (M.O.M.) blocking reagent (Vector) was used to block endogenous mouse antibody in the tissue section (when a primary antibody was raised in mouse) or bovine serum albumin was used to block unspecific binding of antibodies (when an antibody was not in other specie than mouse). Slides were incubated with primary antibodies: Pcp2 (1:100; sc-49072), Zebrin II (1:50; ab115212), Iba1 (1:400; ab5076), ASC (1:400; Al177), Casp-3 (1:100; ab2302), Hsp70 (1:100; sc-66048), Ub (1:100; ab7780), and HA (1:100; ab9110), followed by incubation with an AF488, AF594, or AF647-labeled donkey anti-mouse, anti-rabbit or anti-goat IgG antibody (1:250; Invitrogen), and counterstained with DAPI (Sigma Aldrich). Images were captured using Zeiss LSM 710 upright confocal microscope as Z-stacks and presented as the sum of the Z-projection. The number of Pcp2-labeled or Iba1-labeled cells was determined separately in every visible lobule of the vermis and in the hemispheres. For each mouse, three nonadjacent sections (separated by 70 μm) from the region of vermis were analyzed and the mean value was recorded. Total of five mutant and five control animals was included in each of the studies.