Goat anti guinea pig igg

Ovaries were dissected in PBS, fixed in 4% PFA in PBS and followed by immunofluorescence staining protocol for incubation of primary antibodies. After washing, the manufacturer's protocol (Sigma-Aldrich/Thermo Fisher USA) modified for whole-mount tissue staining was followed (Wang et al. 2015 (link)). Goat anti-guinea pig IgG (Invitrogen, USA) was conjugated with Plus oligonucleotide following the manufacturer's protocol (Duolink in situ Probemaker Plus, Sigma-Aldrich/Thermo Fisher USA). Single slice of confocal image was used. For quantification of proximity ligation assay (PLA) data, punctate staining signals were counted.

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.

Synaptic density of cultured neurons was measured by counting PSD95 and vGlut1-labelled clusters attaching to neuronal dendrites and presented as the number of synapses per micron of dendrite as previously described67 (link). Briefly, after fixation in 4% paraformaldehyde for 30 min, neurons were blocked in 5% goat serum for 30 min at room temperature. PSD95, vGlut1 and MAP2 were labelled by rabbit anti-PSD95 (Cell Signaling, #3450, 1:200), guinea pig anti-vGlut1 (Synaptic System, #135304, 1:400) and mouse anti-MAP2 (Sigma-Aldrich, M4403, 1:400) followed by goat anti-rabbit IgG conjugated with Alexa 594 (Invitrogen, A11037, 1:500), goat anti-guinea pig IgG conjugated with Alexa 647 (Invitrogen, A21450, 1:500) and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A11029, 1:500), respectively. Images were collected under a Nikon confocal microscope followed by three-dimensional reconstruction by using Nikon-Elements advanced Research software68 (link). The synapses were defined by colocalization of vGlut1 and PSD95. Dendritic segments between 20 and 50 μm from the soma were used for the analysis69 (link).