Autofluorescence quenching kit

RV specimens fixed with 10% formalin were frozen in melting isopentane. 10 μm cryosections were treated with antigen retrieval buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween 20, pH 9.0) and incubated at 95^°C for 5 min. Samples were then permeabilized with 1% Triton X-100 in PBS for 5 min. Sections were washed/blocked with 5% goat serum in PBS and then incubated with primary antibody for 48 h at 4^°C. The tissues were then washed/blocked with 5% goat serum and incubated with secondary antibody at 37^°C for 30 min. For RV cardiomyocyte imaging, formalin fixed RV specimens were manually dissected to isolate fiber bundles. Antigen retrieval was performed by incubating samples in antigen retrieval buffer at 95^°C for 45 min. Fibers were then permeabilized with 1% Triton X-100 in PBS for 5 min. Cells were then treated with primary antibodies as described above. All samples were embedded in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). RV cardiomyocytes stained for GLO1 were treated with autofluorescence quenching kit (Vector Laboratories) to reduce mitochondrial autofluorescence. Z-stacks of RV sections and cardiomyocyte bundles were obtained with an Olympus FV1000 BX2 upright confocal microscope (Tokyo, Japan) at the University of Minnesota Imaging Center. All images used to compare fluorescence intensity between groups were collected under identical settings.

The pancreas tissues after fixation with 4% paraformaldehyde for 2 weeks were embedded in paraffin, and 5μm sections were stained by hematoxylin-eosin. For the immunofluorescence histochemistry, the cryoprotected pancreas tissues embedded in the OCT medium (Sakura Finetek, Japan) were cut by cryotome (Tissue-Tek® Polar®, Sakura, Japan), and 5μm sections were immersed with heated 0.01% Citrate retrieval buffer to induce epitope retrieval. Non-specific staining was blocked with 1% bovine serum albumin (Nacalai tesque, Japan), and were incubated overnight at 4°C with the primary antibodies at the dilution of 1:100. We used mouse monoclonal anti-human Hsp70 (BD Bioscience, USA), rabbit anti-human activated μ-calpain (order made by PEPTIDE Institute, Japan), rabbit anti-human cathepsin B (Cell Signaling, USA), and mouse monoclonal anti-Lamp2 (Abcam, USA) antibodies. After washings, the sections were incubated for 30 min with secondary antibodies; Alexa Fluor^TM 594 goat anti-mouse IgG [H+L] (Invitrogen, USA), or Alexa Fluor^TM 488 goat anti-rabbit IgG (Invitrogen, USA) at the dilution of 1:500. To block autofluorescent staining, Autofluorescence Quenching Kit (Vector Laboratories, USA) was utilized. The immunoreactivity was observed with the laser confocal microscope (LSM5 PASCAL, Software ZEN 2009, Carls Zeiss, Germany).