M o m mouse igg blocking reagent

Paraffin-embedded sections of testes were deparaffinized and hydrated through xylenes and graded alcohol series. To increase the immunoreactivity, the sections were boiled in 10 mM citrate buffer (pH, 6.1 Bio-Optica, Milan, Italy) in a microwave at 720 W (3 cycles/3 min each). The sections were then subjected to treatment for blocking endogenous peroxidase activity (Dako). After thorough washing, sections were incubated with MOM mouse IgG blocking reagent overnight at 4 °C (Vector Laboratories) according to the manufacturer’s protocol. Then, sections were incubated with rabbit polyclonal to 4-HNE (4 Hydroxynonenal, ab46545, Abcam, 1:100) diluted in MOM diluent for 30 min, according to the Vector Laboratories instructions. 4-HNE was revealed by biotinylated anti-rabbit IgG followed by streptavidin-HRP, DAB substrate buffer and DAB (Dako kit), according to manufacturer’s instructions. Counterstaining was performed with hematoxylin (Bio-Optica, Milan, Italy). Negative controls were performed by omitting primary antibody and substituting it with MOM diluent alone. Finally, sections were dehydrated and mounted with Neomount (Merck, Darmstadt, Germany). They were observed and photographed under a Leitz Laborlux S microscope (Leica, Wetzler, Germany) equipped with an Olympus digital compact camera.

Coronal brain sections (30 μm) from mice receiving rAAV2-eGFP and Drp1-K38A-eGFP were incubated in M.O.M mouse IgG blocking reagent (Vector Laboratories) overnight before incubation with polyclonal anti-GFP (1:500, Invitrogen) and monoclonal anti-TH (1:500; Sigma). For Fis1-myc, monoclonal anti-myc (1:2,000, 9B11 clone, Cell Signaling) and polyclonal anti-TH (1:500, Calbiochem) were used. Corresponding secondary antibodies Alexa Fluor 488 and 594 (Invitrogen) were used. Nuclei were visualized with 4′,6-diamidino-2-phenylindole dihydrochloride (1:1,000, Invitrogen). Images were scanned at 0.5 μm intervals throughout the whole section and analysed using confocal microscopy (FV1000; Olympus).