*5.3. Expression of Adhesion Molecules*

5.3.1. Evaluation of Protein Expression in Endothelial Cells of the Cremaster Muscle Using Immunohistochemistry

Adhesion molecules expression was assessed 2, 4, or 24 h after the subcutaneous administration of toxins (0.5 μg/100 μL) or 100 μL of phosphate-buffered saline (PBS, as a negative control) in the scrotum (*n* = 5/group). For the immunohistochemical study, animals were anesthetized and transcardiacally perfused with PBS followed by a fixative solution comprising 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). After perfusion, the cremaster muscle was collected, postfixed with 4% PFA for 6 h, and then transferred to cryoprotectant solution of 30% sucrose in PB for 48 h. The cremaster muscle was incubated with anti-ICAM-1 and anti-PECAM-1 primary antibodies (1:100, Biosciences Product) diluted in 0.3% Triton X-100 for 48 h at room temperature with constant agitation. The tissue was washed with PBS (3 times/10 min) and incubated with the secondary antibody (FITC-conjugated AffiniPure goat anti-mouse IgG and TRICTconjugated AffiniPure, Jackson ImmunoResearch Laboratories; diluted 1:50 in 0.3% Triton X-100) for 2 h at room temperature. After three washes with PBS, the tissues were mounted on slides with Vectashield (Vector Labs, Burlingame, CA, USA) [30]. The tissues were stored at 4 ◦C, and the fluorescence was measured using a Zeiss confocal microscope (Zeiss LSM Image Browser program version 4.0.0.157).

5.3.2. Evaluation of Protein Expression in Endothelial Cells of the Cremaster Muscle Using Indirect ELISA Assays

Adhesion molecules expression was also evaluated using indirect ELISA assays [54] after the same treatment described for the immunohistochemical assay. The cremaster muscle was removed, placed in 200 μL of 1 M Trisma solution (pH 7.5) containing protease inhibitors (EDTA 1 mM, PMSF 2 mM and aprotinin 0.3 μM), and disrupted with an ultrasonic homogenizer. The homogenate was centrifuged at 12,000× *g* for 20 min at 4 ◦C, and the protein present in the supernatant was quantified using the Bradford method. ELISA microplate wells were coated overnight at 4 ◦C with 50 μg of protein diluted in 100 mM carbonate-bicarbonate buffer (pH 9.6). The plates were washed three times with PBS-Tween (0.05%) and blocked with 1% bovine serum albumin (BSA) for 1 h at 37 ◦C.

The plates were washed again and incubated overnight at 4 ◦C with anti-ICAM-1 and anti-PECAM-1 antibodies (1:500, Biosciences Product). Then, after a new series of three washes, the plates were incubated for 2 h at 37 ◦C with anti-IgG conjugated with peroxidase (1:2000 —Jackson ImmunoResearch Laboratories). The antibodies were diluted in 1% BSA/PBS-Tween 0.05%. The reaction was visualized using 0.7 mg of OPD (o-phenylenediamine) diluted in 100 mM phosphate-citrate buffer (pH 5.5) containing 0.05% H2O2, stopped after 10 min by adding 50 μL/well of 8 N H2SO4, and quantified by reading the absorbance at 492 nm using an ELISA plate reader (Multiskan EX Thermo Scientific). All ELISA experiments were performed in triplicate in three independent experiments.

### *5.4. Real-Time PCR Analysis of Gene Expression in Endothelial Cells of the Cremaster Muscle*

The expression of adhesion molecule transcripts was measured 30 min, 2 h, and 6 h after the subcutaneous administration of toxins (0.5 μg/100 μL) or PBS (100 mL) as a negative control into the scrotum. Total RNA was extracted from the dissected cremaster muscle in TRIzol solution (Invitrogen) using an ultrasonic homogenizer. Five micrograms of total RNA were reverse transcribed into cDNAs using Superscript III RT (Invitrogen). Quantitative real-time PCR was performed in a Line Gene K Thermal Cycler (Hangzhou Bioer Technology Co., Hangzhou, China) using a Platinum Syber Green qPCR Supermix-UDG kit (Invitrogen) with the following thermal cycling protocol: 15 min at 95 ◦C followed by 40 cycles of 15 s at 95 ◦C, 30 s at 58 ◦C, and 30 s at 72 ◦C. All procedures were performed according to the manufacturer's instructions. The primer sequences for mouse β-actin (FW: 5 CCCAGGCATTGCTGACAGG3 ; RV: 5 TGGAAGGTGGACAGTGAGGC3 ) were designed using sequence alignments obtained from NIH/NCBI based on the published RNA sequence. The primer sequences for mouse ICAM-1 (FW: 5 AAGGAGATCACATTCACGGT3 ; RV: 5 GCCTCGGAGACATTAGAGAA3 ) and PECAM-1 (FW: 5 AGAGACGGTCTTGTCGCAGTA3 ; RV: 5 CGCACACCTGGATCGG3 ) were obtained from the literature [55]. The relative mRNA expression level was determined using the 2−ΔΔCt (cycle threshold) method, and data were normalized to β-actin mRNA expression levels. All real-time PCR experiments were performed in triplicate using samples from three independent animals.

#### *5.5. Integrin Expression in Peritoneal Leucocytes*

The expression of CD11a and CD11b integrins in leukocytes that migrated to the peritoneal cavity in response to toxin injection was evaluated using flow cytometry. For this experiment, the mice were injected (i.p.) with toxins (2.0 μg/300 μL) or sterile PBS and sacrificed in a CO2 chamber 4 h later. Their peritoneal cavities were washed with 5.0 mL of PBS. The peritoneal fluid was collected and centrifuged (1000× *g*/10 min at 4 ◦C), and the precipitate was resuspended in red blood cell lysis buffer and centrifuged (1000× *g*/10 min at 4 ◦C). The pellet was resuspended in 1 mL of PBS. The total leukocyte count was determined using an automatic veterinary hematology analyzer (Mindray BC 2800 VET). The peritoneal cells from different animals were pooled to obtain a concentration of 106 cells/mL. Cells were incubated with anti-mouse FcγRII/RIII at a concentration of 1 μg/106 cells (BD System) for 30 min at 4 ◦C followed by washes with PBS containing 1% serum albumin (BSA) and centrifugation (300× *g*/10 min). The expression of different molecules was analyzed by incubating the cells with 1:200 dilutions of Fluorescein (FITC) conjugated anti-mouse CD11a and CD11b antibodies for 30 min at 4 ◦C. All cell suspensions were also incubated with the respective fluorescein-labeled isotype control monoclonal antibodies (e-Bioscience). The cells were washed and resuspended in PBS containing 0.1% paraformaldehyde. The flow cytometry analyses (10<sup>4</sup> events per data acquisition file) were performed with FACSCalibur using Cell Quest software (Becton Dickinson). All flow cytometry experiments were performed in triplicate in three independent experiments.
