The sections were fixed using cold acetone, blocked using 10% horse serum, and then incubated with rabbit anti-laminin antibody (Sigma, Saint Louis, MO, USA) at a dilution of 1∶400.
Following a 48 hr knock-down period, cells were fixed using 4% paraformaldehyde, blocked with 3% BSA, and either directly mounted onto slides using hard-setting Vectashield (containing DAPI) or first stained with Rhodamine Phalloidin (Biotium, Inc., Cambridge, UK), before mounting.
Adherent cells were fixed using 4% PFA, permeabilised with 0.5% Triton-X100 (Sigma) and blocked with 3% BSA (Sigma)/PBS for 60 min at room temperature.
Cells grown on a microscope coverglass (Thermo Fisher Scientific) were fixed using 4% p-formaldehyde, permeabilized using 0.1% Triton X-100, and blocked in 1% casein.
Harvested organs were fixed using 4% PFA dissolved in PBS at 4°C overnight and embedded in paraffin blocks.
Cells were fixed using Cytofix Cell Fixative BD Biosciencess).
Cells were fixed using 4% paraformaldehyde in PBS and then permeablised using 0.25% Triton X-100 in PBS before blocking with 3% bovine serum albumin in PBS.
Cryosections of soleus and EDL muscles, frozen as a single block, were fixed in 4% paraformaldehyde and permeabilized using 0.2% Triton X-100 prior to staining with antibodies anti-Idh2 (HPand7831) and Idh3a (AV42237), both polyclonals from SIGMA.
As with pure blocks, the order of mixed blocks was fixed: neutral-emotional-neutral-emotional.
A plexiglas platform is used, on which two thick Cu blocks are fixed.
