After dye extravasation was sketched, rats were fixed with 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde via transcardial perfusion before removing the area of skin with the extravasated EB. Then the EB extravasated skin and its control area within a 2 mm radius from the extravasated dots, as well as the corresponding skin areas in saline treated rats, were collected. Following perfusion–fixation, the collected tissues were post-fixed in 4% paraformaldehyde at 4 °C for 4 h and cryoprotected in phosphate-buffered 20% sucrose at 4 °C for 24 h. Following post-fixation, the skin was embedded in artificial medium (Shandon Cryomatrix™, 120 mL, Thermo Scientific, USA), frozen, and cut into 30 μm sections. The sections were then thaw-mounted on SuperFrost® Plus slides (Thermo Scientific, USA) and allowed to dry. Skin sections from HCl- and saline-treated rats were mounted on the same slide to guarantee equal incubation conditions. After an initial wash in 0.1 M PBS (pH 7.4) tissues were preincubated in a solution of 3% normal goat serum and 0.5% Triton X-100 in 0.1 M phosphate-buffered solution (PB, pH 7.4) for 30 min to block non-specific binding. Antibodies were diluted in a solution containing the same substances. Sections were then incubated for 24 h at 4 °C. Immune-fluorescent staining for CGRP, SP, 5-HT or HA was conducted by using specific primary antibodies (Table
1) from different species, mouse and rabbit, which were incubated with the sections simultaneously. After washing in 0.1 M PBS (3–10 min), tissue was exposed to Alex a Fluor 488 goat anti-mouse (1:500; A11001, 0.5 mL, Invitrogen, USA) or Alexa Fluor 488 goat anti-rabbit (1:500; A11008, 0.5 mL, Invitrogen, USA) IgG secondary antibody for 2 h and washed with 0.1MPB. The tissue was then counterstained with blue-fluorescent DAPI nuclei acid (1/40,000, D3571, 10 mg, Invitrogen, USA) for 5 min to label cell nuclei. Following a final wash in 0.1 M PBS, slides were coverslipped with PBS–glycerol. Negative controls were performed by leaving out the primary antibodies during the staining procedure. Mast cells were labeled with the anti-mast cell tryptase antibody and, simultaneously, by HA or 5-HT primary antibodies to observe the coexpression, then were also counterstained with DAPI for nucleus labeling. The secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (c; A11008, 0.5 mL, Invitrogen, USA) or Alexa Fluor 594 goat anti-mouse IgG secondary antibody (1:500; A11005, 0.5 mL, Invitrogen, USA). Slides were recorded with confocal imaging system (FV1200, Olympus, Japan) and analyzed by the Olympus Image Processing Software by an investigator who was blinded to group. Approximately 20 randomized sections from each group were counted for the number of stained cells, the sum length of positive nerve fibers, and the sum fluorescent intensity. All immunohistochemistry for each staining combination was performed at the same time to ensure staining consistency.
Table 1
Primary antibodies for immunohistochemistry
Anti-CGRP Anti-Substance P Anti-Serotonin Anti-Histamine Anti-Mast Cell Tryptase | Rat Rat Rat Rat Rat | 1/1000 1/1000 1/500 1/500 1/1000 | Mouse Rabbit Rabbit Rabbit Mouse | ab81887 ab48326 ab10385 ab123982 ab2378 | Abcam Abcam Abcam Abcam Abcam |