Rabbit anti cd300e polyclonal antibody

Formalin-fixed paraffin embedded tissue sections were stained with a rabbit anti-CD300E polyclonal antibody (Sigma-Aldrich). On appropriate antigen retrieval (water bath at 98°C for 40 min in ethylenediaminetetraacetic buffer pH 8.0), reactivity was revealed using NovoLink Polymer horseradish peroxidase-linked (Leica Biosystems) followed by diaminobenzidine. Characterization of CD300E positive cells was performed by double immunohistochemistry. After completing the first immune reaction, the second was realized using a monoclonal primary antibody to CD163 (clone 10D6, ThermoFisher), visualized using Mach 4-AP (Biocare Medical), followed by Ferangi Blue (Biocare Medical) as chromogen. Quantification of CD300E-expressing cells was performed on at least five high-power fields on sections double stained for CD300E and CD163. Immunostained sections were photographed using the DP-70 Olympus digital camera mounted on the Olympus BX60 microscope, and the digital pictures (each corresponding to 0.036 mm²) were used for cell count. Values were expressed as the mean ± SEM.

Immunohistochemistry was performed as reported elsewhere^{7 (link)}. Briefly, formalin-fixed paraffin embedded tissue sections were stained with a rabbit anti-CD300e polyclonal antibody (Sigma-Aldrich). On appropriate antigen retrieval (water bath at 98 °C for 40 min in ethylenediaminetetraacetic buffer pH 8.0), reactivity was revealed using NovoLink Polymer horseradish peroxidase-linked (Leica Biosystems, Wetzlar, Germany) followed by Diaminobenzidine (DAB). Analysis of CD300e positive cells was performed by double immunohistochemistry. After completing the first immune reaction, the second was realized using a monoclonal primary antibody to CD163 (clone 10D6, ThermoFisher), visualized using Mach 4-AP (Biocare Medical, Pacheco, CA, USA), followed by Ferangi Blue (Biocare Medical) as chromogen. Quantification of positive cells was performed on 10 high power fields (HPF) on sections double stained for CD300e and CD163. Immunostained sections were photographed using the DP-70 Olympus digital camera mounted on the Olympus BX60 microscope, and the digital pictures (each corresponding to 0.13 mm²) were used for cell count. Values were expressed as mean percentage of CD300e positive cells inside the CD163 positive cells. Based on the intensity of CD300e expression, CD163 positive cells were classified as negative, CD300e^bright and CD300e^dim.

After 24 and 48 h from Hp infection, macrophages were lysed in Triton X-100 lysis buffer (20 (link)), and proteins were quantified by BCA protein assay kit (ThermoFisher), according to the manufacturer’s instructions. Equal amounts of proteins were resuspended in NuPAGE LDS sample buffer (Novex, Life Technologies) supplemented with 50 mM of DTT and denaturated for 5 min at 100°C. Samples were separated electrophoretically in NuPAGE Bis-Tris 4–12% polyacrylamide gel (Novex, Life Technologies), and proteins were subsequently transferred onto PVDF membranes (Amersham). Membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS, 50 mM Tris–HCl pH 7.6, 150 mM NaCl) containing 0.1% Tween20^® (Sigma-Aldrich), and antigens were revealed using a rabbit anti-CD300E polyclonal antibody (1:200, Sigma-Aldrich) and a monoclonal anti-HSP90 antibody (1:10,000, Origene). Blots were washed three times with TBS plus 0.1% Tween20^® and incubated for 1 h at RT with horseradish peroxidase-conjugated anti-rabbit (Millipore) or anti-mouse IgG secondary antibody (Novex, Life Technologies), respectively. Blots were developed with enhanced chemiluminescence substrate (EuroClone), and the protein bands were detected using ImageQuant™ LAS 4000 (GE Healthcare Life Science).