Rabbit anti cd4

For histologic examination, mice were intra-dermally challenged five days after the last sensitization. After 20 min, 24 h, and 48 h, the mice were sacrificed at the indicated time and the abdominal skins from the challenge sites were removed and placed in 10% formalin overnight at room temperature. The removed skin samples were 2 mm larger than the lesions, and sized varied from 4–7 mm in diameters (16–49 mm²). Briefly, the tissues were embedded in paraffin, cut into 5-μm sections, de-paraffinized, dehydrated, and stained with H and E. Moreover, sections were further stained with rabbit anti-CD4 (1∶800 dilution) or anti-CD8 (1∶400 dilution) polyclonal antibodies (Bioss, MA, USA). To detect CD marker positive cells, the sections were incubated with peroxidase-conjugated goat anti-rabbit IgG and stained in a substrate solution containing DAB in Bond automatic system (Leica, Newcastle, UK). Inflammatory cell infiltrates were examined by light microscopy and corresponding images were shot by the Olympus BX51 microscopic/DP71 Digital Camera System (Nagano, Japan).

Paraffin-embedded tumor tissues were used in the immunohistochemical staining of CD4⁺ and CD8⁺ T cells. A microtome (Leica, Wetzlar, DEU) was used to prepare the 4-µm-thick tissue sections before deparaffinization and rehydration. Antigen epitopes were retrieved by autoclaving at 121 °C for 20 min in 10 mM sodium citrate buffer (pH 6.0). The sections were incubated overnight at 4 °C with the rabbit anti-CD4 (Bioss, Boston, MA) or anti-CD8 (Abcam, Cambridge, UK) antibody that was diluted 1:100. Subsequently, the sections were incubated for 60 min at room temperature with the secondary antibody HRP-conjugated anti-rabbit IgG (H + L) (Promega, Madison, WI). Positive signals were visualized by using 3,3’-diaminobenzidine (Sigma-Aldrich) for 3 min before the sections were counterstained with hematoxylin for 5 min. As negative controls, the sections were stained in parallel with isotype control, the rabbit monoclonal IgG antibody (Abcam). CD4- or CD8-positive cells were counted as described previously [11 (link)].

We further verified the expression levels of SLC35A3 and CD4 proteins in tumor tissues and adjacent normal tissues of colorectal cancer patients through IHC. After deparaffinization, rehydration, and antigen retrieval by heating from the slides in sodium citrate buffer (pH 6.0), the antigens were retrieved. To inhibit endogenous peroxidase activity, they were blocked in 3% hydrogen peroxide for half an hour and then washed three times with PBS. Incubation with rabbit anti-SLC35A3 (SinoBiological, China) and rabbit anti-CD4 (Bioss, China) was performed overnight at 4 °C, followed by incubation with goat anti-rabbit IgG (TransGen Biotech, China) for 2 h at room temperature. After staining with 3,3′-diaminobenzidine (DAB), the sections were counterstained with hematoxylin, dehydrated, fixed, and sealed with a cover slip. The immunohistochemical results were analyzed in Image-Pro Plus 6.0, using the same brownish-yellow as the uniform standard for judging whether the image was positive. The cumulative optical density (IOD) and pixel area (area) of the tissue in each positive standard photo were analyzed, and the mean density was calculated as Mean density = IOD/area. The higher the Mean density value, the higher the expression level of the positive protein.