Alexafluor 568 goat anti guinea pig igg

For colocalization studies, paraffin sections were treated with 0.05% pronase for antigen retrieval, blocked with 2% bovine serum albumin (BSA; Sigma)/phosphate buffered saline (PBS), incubated overnight (4° C) with 10E4 (anti-HS) mAb (1/10), washed and stained with AlexaFluor 488-goat anti-mouse IgM (Thermo Fisher, Rockford, IL, USA). The same sections were washed, incubated with rabbit anti-human glucagon IgG (Abcam, Cambridge, UK) or guinea-pig anti-insulin Ig (Dako, Santa Clara, CA, USA), washed and stained with Alexafluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher) (https://dx.doi.org/10.17504/protocols.io.kvycw7w). The specificity of HS staining was checked on serial sections using IgM_κ isotype control (BD Biosciences), instead of 10E4 mAb, together with anti-glucagon or anti-insulin antibody. Nuclei were stained with DAPI (0.2 μg/ml; Sigma). Sections were imaged using an automated Axio Observer inverted fluorescence microscope (Zeiss; Göttingen, Germany). Merged images were prepared using ZEN (version 2.3) software (Zeiss).

For colocalization studies of COL18, paraffin sections were treated with 0.05% pronase for antigen retrieval, blocked with M.O.M Ig block in 2% bovine serum albumin (BSA; Sigma)/phosphate buffered saline (PBS) and incubated overnight (4° C) with COL18A1 mAb (1/50). After washing, the sections were then stained with AlexaFluor 488-donkey anti-mouse IgG (Thermo Fisher, Rockford, IL, USA). The sections were washed and then incubated with rabbit anti-human glucagon IgG (Abcam, Cambridge, UK) or guinea-pig anti-insulin Ig (Dako, Santa Clara, CA, USA). Following washing, the sections were stained with AlexaFluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher) (https://dx.doi.org/10.17504/protocols.io.btqynmxw). Background staining was determined using sections stained only with the secondary antibody. Nuclei were stained with DAPI (0.2 μg/ml; Sigma). After washing, Trueblack (Biotium, Fremont, CA; 1/20 in 70% ethanol) was applied for 30 secs to reduce autofluorescence. The slides were washed and mounted using a 1.5H cover glass (Marienfeld, #0107222, Lauda-Konigshofen, Germany) and antifade mounting medium (ProLong Diamond, Invitrogen). Sections were imaged using an automated Axio Observer inverted fluorescence microscope (Zeiss; Göttingen, Germany). Merged images were prepared using ZEN (version 2.3) software (Zeiss).