Streptavidin hrp

Mice were euthanized 8 days following infection and the first 10 cm of the duodenum and jejunum were flushed with PBS, fixed in 4% paraformaldehyde for 4 hours, washed in PBS overnight, and placed in 30% sucrose for 6 hours before embedding in OCT medium. Eight micron cryosections for both DCLK and MUC2 staining were cut and placed on slides for histology. For detection of DCLK⁺ cells, slides were incubated with: 1% H₂O₂ in PBS for 45 minutes, TNB and Fc block for 30 minutes, and biotin/avidin for 30 minutes each. Tissues were stained with 1:1000 rabbit anti-DCLK (Abcam 31704) in TNB followed by 1:500 biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch), streptavidin-HRP (Perkin Elmer), 1:200 555-tyramide (Perkin Elmer), and DAPI and mounted with a coverslip (Vectashield). For detection of MUC2⁺ cells, tissues were blocked with 5% BSA and Fc block and stained with 1:200 anti-mouse MUC2 (Santa Cruz H-300), followed by goat anti-rabbit 647 (LifeTechnologies), and DAPI. Images were obtained on a Marianas microscope. Images were blinded and the number of DCLK⁺ cells/mm villus were quantified using Fiji (ImageJ) where only intact villi were assessed. A similar blinded quantification was used for MUC2 staining but since mucin is released from the cell, the number of MUC2⁺ foci/mm villus was assessed by enumerating only the regions where discrete MUC2 staining originates.

Transfected HEK293 cells on glass coverslips were rinsed 24 hours following transfection two times in prewarmed phosphate buffered saline (PBS), fresh growth media supplemented with 50 μM biotin was added and the cells incubated overnight at 37°C. The following day, the cells were rinsed two times in PBS, fixed in 4% paraformaldehyde in PBS and permeabilized in 0.25% Triton X-100/PBS for 5 minutes. Cells were then rinsed in PBS and blocked in immunoblock buffer (2% goat serum, 3% bovine serum albumin, 1X PBS) before the addition of antibodies (1:400 mouse anti-HA (Thermo Fisher Scientific) or Streptavidin-HRP (Genscript, Piscataway, NJ, USA)) and incubated overnight at 4°C. Secondary antibody was used to visualize the HA epitope (1:50 goat anti-mouse Alexa 594 (Thermo Fisher Scientific)). To amplify the Streptavidin-HRP signal, tyramide signal amplification was used (TSA-FITC) according to manufacturer’s instructions (PerkinElmer). DAPI (Thermo Fisher Scientific) was added to all coverslips to visualize cell nuclei. Coverslips were rinsed and mounted in Fluoromount G (Electron Microscopy Science, Hatfield, PA, USA) and imaged on a Nikon TiE inverted epifluorescence microscope equipped with a 100x/1.4NA Plan-Apochromat VC oil immersion objective, a Lumencor SOLA LED light source for epifluorescence illumination, and an Andor iXon Ultra 897 EMCCD camera.