Rabbit anti human cathepsin b

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).