Ck nac liquiform

Myonecrosis was evaluated by quantifying the plasma creatine kinase (CK) activity according to the instructions for the CK-NAC Liquiform (LabTest, Brazil). Each mouse received an i.m. injection of 40 μg of BaV in 40 μL of saline into the gastrocnemius muscle. At 3 and 24 h after the venom injection, blood samples were collected by an orbital puncture for determination of the serum CK levels, which were expressed as units/L.

3,3′,5,5′-Tetramethylbenzidine (TMB), bovine serum albumine (BSA), ATP, protease and phosphatase inhibitors, hydrogen peroxide, bicinchoninic acid (BCA) protein assay kit, anti-β-actina (A1978-200UL) isotype mouse, and 3,3′diaminobenzidine (DAB) were purchased from Sigma Aldrich (Sant Louis, MO, USA). Goat anti-mouse antibody conjugated to horseradish peroxidase (A90-116P) was purchased from Bethyl Laboratories (Montgomery, TX, USA). Mouse IL-1β/IL-1F2 kit DuoSet ELISA was purchased from R&D Systems (Oxon, UK). Anti-caspase-1 p10 (AG-20B-0044-C100) and anti-NLRP3 (AG-20B-0014), both from mouse isotype, were purchased from Adipogen Life Sciences (San Diego, CA, USA). PVDF Membrane was purchased from Millipore (Darstadt, Germany). P2X7 receptor antagonist and A438079 were purchased from Tocris Bioscience (Bristol, UK). CK-NAC Liquiform and LDH Liquiform were purchased from Labtest (Minas Gerais, Brazil). Reagents were obtained from Merck (Darmstadt, Germany). Trizol^® Reagent and SuperScript III Reverse Transcriptase were purchased from Thermo Fisher Scientific (Waltham, MA, USA). iTaq Universal SYBR® Green Supermix was purchased from Bio-Rad (Hercules, CA, USA).

Muscle regeneration was evaluated by the quantification of the muscle tissue creatine kinase (residual CK) activity and by histological analysis. Each mouse received an i.m. injection of 40 μg of BaV in 40 μL of saline into the right gastrocnemius muscle and the same volume of saline into the left gastrocnemius muscle followed by the treatment protocols. Groups of mice were euthanized 1, 4, 7, 14, 21 and 28 days after the venom injection, and both gastrocnemius muscles were collected, weighed, and homogenized in 4 mL phosphate buffered saline (pH 7.4) containing 0.1% Triton X-100. After the centrifugation (2100 g/5 minutes), the supernatant was collected for determination of the residual CK (CK-NAC Liquiform, LabTest). The results are expressed as the percentage of CK obtained in the venom-muscle injected (residual CK) compared with the CK content of the control contralateral muscle.
In another set of experiments, the injected muscles were collected for histological analysis. They were fixed in Bouin’s solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin (HE) for light microscopic analysis.