Guinea pig anti swine insulin

Kidneys were immersed in Tissue-Tek OCT (Bayer) and quick frozen on dry ice. Using cryomicrotome and Superfrost Plus slides (Fisher Scientific), 6-μm tissue sections were cut. Sections were fixed with 100% acetone at room temperature, and after washing in TBS, an avidin/biotin-blocking step was included (Vector Laboratories). Primary and secondary antibodies (Vector Laboratories) reacted with the sections for 60 min each, and colour reaction was obtained by sequential incubation with Vector Blue AP III and AEC (Vector Laboratories) as previously described [38 (link)]. Rat anti—mouse CD8a (Ly2)-biotin, rat anti—mouse CD4 (L3T4; BD Biosciences)-biotin and guinea pig anti-swine insulin (DAKO) were used. Goat anti-guinea pig AP was used to detect insulin [38 (link)].

The paraffin sections were stained as previously described [12] (link). Antibodies used were guinea pig anti-swine insulin (Dako Cytomation, Glostrup, Denmark), mouse anti-BrdU (Dako Cytomation), rabbit anti-survivin (Cell Signalling Technology), rabbit anti-C-peptide (Cell Signalling Technology, Danvers, MA, USA) and rabbit anti-ChromograninA (Dako Cytomation). Specificity of the survivin antiserum was confirmed by preabsorbtion with a survivin blocking peptide for 30 min (Cell Signalling Technology).
For light microscopy stainings the sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Zymed Laboratories, South San Francisco, CA, USA), rinsed and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories). The sections were finally developed with 3-amino-9-ethyl-carbazole substrate (Thermo Scientific). For fluorescence microscopy, the secondary antibodies used were goat anti-guinea pig IgG (Alexa, A11076, Invitrogen, Paisley, UK), donkey anti-rabbit IgG (Alexa, A21206, Invitrogen) and donkey anti-mouse IgG (Alexa, A21202, Invitrogen). Nuclear staining was performed with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA, USA).

The pancreata of FVB control and pregnant mice were processed as described by Alanentalo et al. [17] (link). Shortly, the pancreata were fixed in 4% PFA for 4 h, dehydrated in methanol, quenched in MeOH: DMSO: H₂O₂ (2∶1∶3), followed by rehydration to TBST and incubations with guinea pig anti-swine insulin (DakoCytomation, Glostrup, Denmark) and goat anti-guinea pig IgG (Alexa, A11076, Invitrogen). The stained pancreata were mounted in low melting agarose, followed by dehydration in methanol and clearing with BABB. Bioptonics 3001 OPT scanner (Bioptonics) was used for the scanning of the samples, with exiter D560/40 nm and emitter 610 nmLP or exiter 425/40 nm and emitter 475 nmLP when visualizing Alexa 594 and 488 respectively. Scanning settings (identical for all specimen) were: rotation degree 0,9 um; pixel size 18,23 um; resolution 1024×1024 pixels; exposure time was set manually depending of the intensity of the fluorescence. All samples were scanned with the same zoom. OPT sample reconstructions were made with NRecon v1.6.3.3 (SkyScan). Isosurface reconstructions were generated using Imaris v 7.6.3 (Bitplane). Islet volumes were segmented using the “background subtraction (local contrast)” thresholding option and the intensity threshold was set manually for each pancreas.