Universal blocking reagent

Immunofluorescent staining was performed as described previously [33 (link),37 (link)]. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature at specific time points. Cells were permeabilized with 0.1% Triton for 10 min at room temperature. Cells were blocked with 1% Universal Blocking Reagent (BioGenex, Fremont, CA, USA) for 30 min at room temperature, and incubated with specific antibodies overnight at 4 °C. The primary antibodies used were 1:200 fibrous-actin (FA) (Invitrogen, Waltham, MA, USA), 1:500 microtubule-associated protein 2 (MAP2) (Biolegend, San Diego, CA, USA), 1:500 beta-III tubulin (TuJ1) (Biolegend, San Diego, CA, USA), and 1:500 glial fibrillary acidic protein (GFAP) (Invitrogen, Waltham, MA, USA), ionized calcium-binding adaptor protein 1 (Iba1) (Fujifilm, Tokyo, Japan). Corresponding secondary antibodies were used the next day: 1:500 goat anti-chicken (Invitrogen, Waltham, MA, USA), 1:500 rabbit anti-mouse (Invitrogen, Waltham, MA, USA), and 1:500 donkey anti-rabbit (Invitrogen, Waltham, MA, USA). Secondary antibodies were added and kept at room temperature for 30 min. After washing, cells were mounted with Prolong™ gold antifade reagent with DAPI (Invitrogen, Waltham, MA, USA). After 24 h, cells were imaged at 10× magnification using Leica DMi8 microscope, obtaining at least five images from different areas in each well.

Antigen retrieval was performed on 5 μm sections of PC3 tumors by heating at 126°C for 11 minutes in Target Retrieval Citrate buffer (pH 6, Dako, Denmark). After blocking endogenous peroxydases and non-specific antigenic sites (Universal Blocking Reagent, Biogenex, USA), sections were incubated with primary anti-CD31 antibodies (diluted 1:150 in BSA/PBS 1%) for 1h, followed by biotinylated secondary antibodies (diluted 1:300) for 30 minutes and with HRP-conjugated streptavidin (diluted 1:500) for 30 minutes. Signal was amplified by an incubation in Biotinylated Tyramide (diluted 1:100, Perkin Elmer, USA) for 10 minutes, followed by additional 30 minutes in HRP-conjugated streptavidin. Staining was performed with AEC+ High Sensitivity Chromogen (Dako, Denmark) followed by a counterstaining with hematoxylin. Stained sections were scanned with a NanoZoomer slide scanner (Hamamatsu Photonics, Japan) and quantification was performed using ImageJ software (National Institute of Health, USA). Tumor vascularization was quantified both as the number of blood vessels per unit surface and as the % of surface covered by blood vessels.

For each condition, 10⁶ WT or R6/1 BMMCs were incubated with IgE (100 ng/mL) for 24 h. Then, the cells were collected and re-suspended in universal blocking reagent (BioGenex, San Ramon, CA) for 5 min at 4 °C. The cells were washed twice with 1× PBS and incubated with anti-FcεRI (1:500 in blocking buffer containing 1× PBS, 5 g/L BSA, 0.5 g/L sodium azide), and anti-TLR-4 N-terminal Ab (1:100 in blocking buffer) for 30 min at 4 °C. The cells were washed and incubated with a PE-coupled anti-IgG Ab (1:200 in blocking buffer). After 30 min of incubation at 4 °C, the cells were washed again and re-suspended in 1% paraformaldehyde (PFA). An anti-isotype staining was performed at the same time. The samples were analyzed in a CytoFLEX LX (B-R-V-Y-N-I) flow cytometer (Beckman Coulter, Brea, CA, USA), using the software CytExpert for data acquisition; plots showing PE⁺ populations were generated with the software Kaluza Analysis. An Attune flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) was used in some experiments.