Anti tryptase

For IHC analyses, brain tissue sections were incubated for 1 h in 10% bovine serum albumin (BSA) with 0.3% Triton X-100 in 0.01 M PBS and then overnight with anti-tryptase (Abcam, UK) at 4°C. Tissue sections were washed and incubated with anti-rabbit secondary antibody for 1 h at room temperature. Immunostaining was visualized with DAB and counterstained with hematoxylin (Solarbio, China). The slides were scanned using a digital camera (Olympus, Japan). For IF analysis, brain sections or cell slides were incubated overnight with primary antibodies: ZO-1, occludin and Ki67 (Abcam, UK) in blocking solution at 4°C, followed by goat anti-mouse Alexa Fluor Plus 488 secondary antibody (Invitrogen, USA), and then covered with mounting medium containing DAPI (Invitrogen, USA) in a dark environment. Images were captured under a fluorescent microscope (Carl Zeiss, Germany). Images were calibrated for the optical density and then measured the areas and integrated densities. Changes in immunofluorescent intensity were shown (mean fluorescence intensity = integrated densities/areas)

The paraffin-embedded tissue sections on the slides were deparaffinized with xylene and dehydrated with gradually decreasing concentrations of ethanol. The dehydrated tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. The tissue sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95°C. The final step was repeated using a 10 mM sodium citrate solution (pH 6.0). The sections stayed in the same solution while cooling for 20 min and then rinsed with PBS. Subsequently, the sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37°C. The primary antibodies were used anti-TNF-α and anti-tryptase (Abcam, Cambridge, MA). After three serial washes with PBS, the signal was visualized using an Envision System (DAKO) for 30 min at 37°C. 3,3′-Diaminobenzidine tetrahydrochloride was used as a coloring reagent, and hematoxylin was used as the counterstain. The slides were viewed with a slide scanner (Pannoramic MIDI) and the integrated optical density was analyzed using the iSolution DT software.

Immunohistochemistry was performed essentially as previously described (33 (link)). Briefly, larvae were fixed for 1 h in 4% paraformaldehyde in phosphate-buffered saline (PBS), then rinsed in PBS + 1% Tween 20 (PBS-Tween), dehydrated in 100% methanol and stored at −20°C until use. The following antibodies were used: rabbit anti-GFP (Invitrogen cat: A11122), anti-MPO (Genetext cat: GTX128379) and anti-tryptase (Abcam cat: ab2378). Quantification of neutrophils, mast and lymphoid cells in the intestine was performed in whole-mount larvae; for this purpose, a region in the midgut was defined (red rectangle in Figure 1). Thirty larvae per condition were analyzed in three different experiments.