Rat anti cd4

Skin samples were embedded in OCT (Tissue Tek) and sectioned to 8 μm. HaCaT cells with AQP3 stable overexpression or knockdown were plated in 24-well plates coated with round coverslips and stimulated with 100ng/ml TNF-α (Cat #300-01, PeproTech) in the presence of calcium. The slides and cells were fixed in 4% formalin for 10 min and washed with PBS 3 times for 10 min. Then we blocked with 5% normal donkey serum in PBS supplemented with 0.3% Triton-X100 for 1 h and incubated with primary antibodies at 4 °C overnight. The next day the appropriate fluorophore-conjugated secondary antibodies were used and 4'-6-diamidino-2-phenylindole (DAPI) was used for nuclei visualization. Following primary antibodies were used: anti-human AQP3 (1:100, Sigma-Aldrich), anti-mouse AQP3 (1:500, Abcam), Rat anti-CD4 (1:100, eBioscience), Rabbit anti-p-p65 (1:200, Cell Signaling), Rabbit anti-p65 (1:100, Cell Signaling). The images were acquired using an Eclipse Ni-U Upright Microscope (Nikon) and 5 randomly selected fields were used to count the positive cells. The number of CD4-positive or nuclear p65-positive cells was counted in at least 3 randomly selected microscopic fields (original magnification, 200×) in each sample to calculate the mean number.

Rabbit polyclonal antibody against mouse EPRAP was raised by immunization with keyhole limpet hemocyanin–conjugated synthetic peptides corresponding to amino-acid residues 244–260, 330–346, and 629–645 of murine EPRAP. For immunohistochemistry of mouse samples, paraffin-embedded sections were routinely stained with one of the following antibodies: rat anti-F4/80 (Abcam, Cambridge, MA, USA), rat anti–Gr-1 (eBioscience, San Diego, CA, USA), rat anti-B220 (eBioscience), rat anti-CD4 (eBioscience), rat anti-CD8 (Abcam), hamster anti-CD11c (eBioscience), mouse anti-NCAM (Abcam), rabbit anti–phospho-NF-κB p105 (Ser933) (Cell Signaling, Boston, MA, USA), rabbit anti–phospho-MEK1/2 (Ser221) (Cell Signaling), or rabbit anti–phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling), or rabbit anti-EPRAP.
For immunohistochemistry of human samples, paraffin-embedded sections were stained with rat anti-CD68 (Abcam), goat anti-FEM1A (Abcam), and rabbit anti–phospho-NF-κB p105 (Ser933), rabbit anti–phospho-MEK1/2 (Ser221), or rabbit anti–phospho-p44/42 MAPK (Thr202/Tyr204) antibodies. Staining with non-immune rat or rabbit IgG served as a negative control for each experiment.

Eight weeks after gamma knife irradiation modeling, mice were anesthetized and transcardially perfused with cold PBS, followed by 4% paraformaldehyde (PFA). Brains were extracted and placed into tubes filled with 4% PFA for 1–2 h and cryopreserved with gradient sucrose (20% and 30% sucrose) at 4°C. After being embedded with OCT molds, the mice brains were frozen at −20°C and cut into sections (30 μm thickness) for further immunostaining. A blocking solution with 5% bovine serum albumin (BSA; MRC, USA) and 0.3% Triton X‐100 (Sigma‐Aldrich, Germany) was used to block and permeabilize the frozen sections. For IF labeling, frozen sections were incubated with rat anti‐CD4 (1:200; eBioscience, USA) and GZMB (1:50; Abcam, USA) at 4°C overnight, followed by appropriate Alexa Fluor‐conjugated secondary antibody (1:500; Jackson, USA) at room temperature for 2 h. The brain slices were mounted with VECTASHIELD Antifade Mounting Medium (Vector, USA) with 4′,6‐diamidino‐2‐phenylindole (DAPI) and imaged by fluorescence microscope (Leica DM6B; Germany).