TITLE: Immunofluorescence staining of Collagen Type XVIII in islet beta cells of formalin-fixed mouse pancreas 
AUTHORS: Sarah Popp, Charmaine Simeonovic

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Paraffin sections of formalin-fixed mouse pancreas 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), incubated overnight (4°C) with COL18A1 mAb (1/50; Santa Cruz), washed and stained with AlexaFluor 488-Donkey anti-mouse IgG (Thermo Fisher). The same sections were washed, incubated with rabbit anti-human glucagon IgG (Abcam) or guinea-pig anti-pig insulin Ig (Dako), washed and stained with Alexafluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher). Background staining was determined using sections stained only with the secondary antibody. Nuclei were stained with DAPI (0.2 μg/ml; Sigma). Trueblack (Biotium) was applied to reduce autofluorescence. Sections were photographed using an automated Axio Observer inverted fluorescence microscope (Zeiss). Merged images were prepared using ZEN (version 2.3) software (Zeiss).</div></div>

[STEPS]
?. Deparaffinize slides in each xylene for 1 min (see Guidelines). Rehydrate slides in graded alcohols beginning in absolute ethanol (10 dips)/ container of absolute ethanol), followed by 90% ethanol (10 dips) and 70% ethanol (10 dips). Wash well in running tap water for 5 min.
?. Wipe around sections using tissue and cover each section with pronase solution (2.5 mg pronase in 5 ml tap water (pH 7) i.e. 0.05% pronase) for antigen retrieval. Return humidified slide tray to 37°C incubator for 10 min.
?. Wash sections with phosphate-buffered saline (PBS), 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash.
?. Block sections with M.O.M Ig block/2% bovine serum albumin (BSA) in PBS at room temperature for 30 min.
?. Tip off block, wipe around sections and apply primary Col18A1 mAb, 4 μg/ml in 2% BSA/PBS, 125 μl/section. Incubate overnight at 4°C in a humidified tray (containing PBS).
?. Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash.
?. Apply secondary donkey anti mouse IgG AF488 (Thermo Fisher #A21202), 1 μg/ml with 2% BSA/PBS), 125 μl/section, and incubate for 30 min at room temperature.
?. Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash.
?. Apply anti-insulin or anti-glucagon pAb:(a) For insulin staining, apply polyclonal guinea pig anti-insulin (DAKO #A0564), 130 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature.(b) For glucagon staining, apply rabbit polyclonal anti-glucagon (Abcam #ab133195), 10 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature.
?. Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash.
?. Apply secondary antibodies for anti-insulin or anti-glucagon pAb:(a) For insulin staining, apply goat anti-guinea pig IgG AF568 (Thermo Fisher #A11075), 10 μg/ml dilution in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature.(b) For glucagon staining, apply donkey anti-rabbit IgG AF568 (Thermo Fisher #A10042), 4 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature.
?. Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash.
?. Stain sections with DAPI (1 mg/ml in water; Sigma #D9524), 0.2 μg/ml in PBS for 2 min.
?. Wash sections with PBS, 3 x, then 1 x 5 min in slide container containing 250 ml PBS with agitation of slides (10 x) at 0, 2.5 min and 5 min.
?. Stain sections with 1x TrueBlack (Biotium #23007) in 70% EtOH, 125 μl/section for 30 sec.
?. Wash sections with PBS, 3 x, then 1 x 5 min in slide container containing 250 ml PBS with agitation of slides (10 x) at 0, 2.5 min and 5 min.
?. Mount slides in ProLong® Diamond Antifade Mountant (Thermo Fisher #P36961), using 1.5H cover glass (uniform thickness (170 microns) and low autofluorescence)
?. Image sections using an automated Axio Observer inverted fluorescence microscope (Zeiss). Prepare merged images using ZEN (version 2.3) software (Zeiss).