Ab2378

The slides were deparaffinized, and heat-induced antigen retrieval was performed using Histofine (Nichirei Biosciences Inc., Tokyo, Japan). Slides were washed with Tris-buffered saline containing Tween 20 (3 × 5 min) and incubated with 10% donkey serum for 60 min. Primary antibodies or FITC-conjugated anti-mouse mast cell tryptase antibodies (1:2000, mouse monoclonal, ab2378; Abcam, Cambridge, UK) were applied to the sections and incubated overnight at 4 °C. The primary antibody used was anti-CRHR1 antibody (1:1000, goat polyclonal, ab77686; Abcam, Cambridge, UK). For double immunofluorescence labeling, we used the primary and FITC-conjugated antibodies described above, and the appropriate secondary antibody (donkey anti-goat IgG) labeled with Alexa Fluor 594 (1:1000, ab150136; Abcam). Following incubation with the antibodies, the tissues were washed with Tris-buffered saline containing Tween 20 (3 × 5 min). The slides were coverslipped with Prolong-Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Rockford, IL, USA). Fluorescent images were obtained at the Osaka City University Graduate School of Medicine Core Imaging Facility. The tissues were examined at 400× magnification using a fluorescence microscope (BX53, Olympus).

The intestinal tissues were fixed with 4% paraformaledehyde, embedded in paraffin and cut into slice. After routinely dewaxing and hydration, the slices were conducted antigen heat retrieval in citrate buffer (0.01 M, pH 6.0), and then blocked with 10% donkey serum (containing 0.3% Triton X-100) for 45 min at room temperature. Sections were incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-mast cell tryptase antibody (1:500, ab2378, Abcam) or rabbit monoclonal anti-PAR2 antibody (1:400, ab180953, Abcam). After washing with phosphate buffer saline (PBS) for 5 min * 3 times, sections were respectively stained with donkey anti-mouse or rabbit Alexa Fluor 488 ((IgG H&L) secondary antibodies (1:300, Invitrogen) for 60 min at room temperature, and then washed with PBS for 5 min * 3 times. Finally, DAPI (1 μg/ml, Beyotime Biotech, China) were used for nuclei staining. Images were viewed and captured using a confocal laser scanning microscope (Nikon, Japan) with excitation wavelength appropriate for Alexa Fluor (488 nm or 594 nm), and analyzed using NIS Elements Viewer Software (Nikon, Japan).

Glioma tissue samples were prepared as 4 μm sections. The slides were deparaffinized, rehydrated in a series of gradient ethanol, and recovered them by heating the tissue at 100°C in citrate buffer for 1 hour. The endogenous peroxidase activity was inhibited with 3% hydrogen peroxide for 10 minutes at room temperature. The slides were treated with primary antibodies overnight at 4°C. After washing with PBS, we then incubated it with goat serum at room temperature for 30 min. Each segment was rinsed with PBS and then incubated with DAB for 5 min. Two pathologists assessed the IHC staining. IHC was performed using antibodies against CD3 (ab16669, Abcam), FOXP3 (ab20034, Abcam), Tryptase (ab2378, Abcam), and CD163 (ab79056, Abcam).

For DAB staining, after antigen retrieval, slides of deparaffinized embedded tissue sections were blocked with 3% BSA containing 0.1% TX100 and 0.1% NaN₃ for 1–2 h at room temperature (RT) with rocking. The primary antibody solution consisted of a 1:200 dilution of the anti-mast cell tryptase antibody ab2378 (Abcam) overnight. On the second day, the slides were treated with a 1:200 dilution of the secondary antibody, goat anti-mouse IgG-HRP (Santa Cruz Sc-2005) for 2 h at RT. The slides were then washed with TBST and incubated with the liquid DAB+ substrate solution of the chromogen system (DAKO) until a brown color was observed. After staining, the tissue was counter-stained with hematoxylin for 30 sec and rinsed under running water for 10 min. The slides were then dipped in 100% alcohol three times and then xylene three times. Finally, photographs were taken with a Nikon microscope.

Paraffin-embedded tonsil, 4 normal peripheral nerves (sural nerve), 23 NF1-DNFs (13 males, 10 females) and 55 sp-DNFs (31 males, 24 females) were included in the immunohistochemical analyses. All DNFs were incubated with neurofibromin (Santa Cruz sc-67) antibodies, while and all DNFs, tonsil and normal sural nerve sections were incubated with KIR2DL5 (Abcam; ab175895) antibodies. Biotinylated secondary rabbit antibodies (Vector Laboratories) were used in combination with Vectastain Elite ABC development and hematoxylin counterstaining. Normal human tonsil was used as the reference control tissue for the KIR2DL5 antibody. Images were acquired on a Nikon Eclipse E600 microscope conjugated with a Nikon Plan Fluor 10x/0.30 DIC L objective and a Leica EC3 camera. Normal human sural nerve, sp-DNF sections, as well as primary cell cultures of normal human Schwann cells and HEK293T cells were analyzed by immunofluorescence using S100β (Millipore CB1040), c-Kit (Millipore; MAB1164), tryptase (Abcam; ab2378), vimentin (DSHB; AMF-17b) and KIR2DL5 (Abcam; ab129751) antibodies conjugated to appropriate Alexa Fluor secondary antibodies. Images were acquired on a Nikon Eclipse TE300 fluorescence microscope conjugated with a Nikon Plan Fluor 20x/0.45 ELWD objective and a Leica DFC3000G camera.