Goat anti rabbit af488

C57Bl/6 mice were injected bilaterally with CTB 488 into the colon as described above. NG were dissected 1 week post injection as described above, dipped in Fast Green (1%, Sigma-Aldrich) to assist with visualization and flash frozen in OCT. 15 um sections of NG were sliced on a cryostat for RNAScope/IHC. Samples were processed and stained with Scn5a, positive or negative control probes according to the manufacturer’s instructions. After in situ hybridization sections were washed three times in wash buffer (1X, ACDBio) and then fixed in 1% PFA in TBS for 10 minutes at 4 C to stabilize the ISH labeling. Samples were next washed three times in TBS-T and incubated in 10% Goat Serum in TBS with 1% BSA for 30 minutes. Samples were stained with anti Alexafluor-488 antibody (1:1000, Thermo-Fisher) for 1 hour in TBS-1% BSA. After primary antibody staining, sections were washed three times for 5 minutes each in TBST and stained with Goat anti rabbit AF488 (1:1000, Thermo-Fisher) in TBS-1% BSA for 30 minutes. Samples were again washed three times for 5 minutes each in TBST and finally mounted in Prolong gold antifade with DAPI (Thermo-Fisher) for imaging. Slides were imaged within 24 hours of mounting on an inverted LSM 880 NLO laser scanning confocal and multiphoton microscope (Zeiss) and images were processed using Image J.

Single-cell suspensions were incubated with Fc Block (Bio X Cell, 2.4G2) and stained with antibodies to surface markers diluted in FWB. Cells were washed in FWB and resuspended in FWB containing DAPI (Roche) and flow cytometric counting beads (CountBright Absolute; Life Technologies) for live cell gating and counts. Cells for intracellular staining were washed in PBS and stained with Violet Live/Dead fixable stain (Life Technologies) according to manufacturer’s instructions. Transcription factor staining was done using the FoxP3/Transcription Factor Staining Buffer Set (eBiosciences) according to manufacturer’s instructions. Cells for DCLK1 staining were fixed in 4% PFA for 2–5 min, washed in FWB, and stained with rabbit anti-DCLK1 (Abcam; ab31704; 1:4000) antibody in perm/wash buffer from the FoxP3 staining buffer kit, followed by F(ab’)2 donkey anti-rabbit IgG-PE (Life Technologies; 1:1000) or goat anti-Rabbit AF488 (Invitrogen; 1:4000) for 10 min. Staining was done on ice. For co-staining of DCLK1 and endogenous IL-25 RFP, PFA fixation was performed for 30 seconds on ice. Samples were analyzed on a LSRFortessa (BD Biosciences) with five lasers (355 nm, 405 nm, 488 nm, 561 nm and 640 nm). Samples were FSC-A/SSC-A gated to exclude debris, FSC-H/FSC-A gated to select single cells, and gated to exclude dead cells. Data were analyzed with FlowJo 9/10 (Treestar/Flowjo).

Immunofluorescence studies were performed with transfected HEK293 as previously described [26 (link)]. HEK293 cells were seeded in Ibidi treat 8-well μ-slides (Ibidi, Martinsried, Germany) 24h prior transfection with Lipofectamine LTX (Invitrogen) and VWF-plasmid-constructs in vector pcDNA3 [5 (link)]. Antibodies used were: rabbit anti-VWF (DAKO, Hamburg, Germany, 1:1,000), mouse anti-Protein Disulfide Isomerase (PDI) (Abcam, Cambridge, UK, 1:500), goat anti-rabbit AF488 (Invitrogen, 1:5,000), goat anti-mouse AF 546 (Invitrogen, 1:5,000). Images were captured at RT with a confocal microscope (TCS SP5, Leica, Wetzlar, Germany). For settings, please refer to legend of Fig. 4.