Formalin

Serum samples were heat inactivated at 56 °C for 30 min and then serially diluted 2-fold in DMEM without FBS. Diluted test sera were incubated with an equal volume of p75 variant at a concentration of 100 PFU/mL, at 37 °C and 5% CO₂ for 1.5 h. Confluent Vero cell monolayer in 24-well plates was incubated with 100 μL of virus–sera mixture at 37 °C with 5% CO₂ for 1 h, after which the inoculum was removed and the cells were overlaid with 1 mL of 1.5% carboxymethyl cellulose (Sigma) in 2× DMEM with 2% heat-inactivated FBS. The plates were incubated at 37 °C with 5% CO₂ for 48 h, fixed with 30% formalin (Himedia, Thane, India) in PBS followed by staining with 0.05% crystal violet solution (Sigma). The number of plaques was counted and the PRNT₉₀ titer was calculated and defined as the dilution of serum required to neutralize 90% or more of the virus infection.

Chemicals Carrageenan and Formalin were obtained from Himedia Laboratories Pvt. Ltd (Mumbai, India). The Test compound Zaltoprofen was obtained from JB Chemicals and Pharmaceuticals Ltd (Mumbai, India) and the standard agent Piroxicam from Medipol
Pharmaceutical India Pvt. Ltd. (New Delhi, India).

Lipopolysaccharide (E. coli 055: B5), SDS, EDTA, hydroxylamine hydrochloride, thiobarbituric acid, RNA later, Bradford reagent, and protease inhibitor cocktail were procured from Sigma-Aldrich (St. Louis, MO, United States). TRIzol was purchased from Invitrogen, California, United States. de Man, Rogosa, and Sharpe (MRS) agar, glass slides, paraffin, hematoxylin, eosin, alcian blue, glycerol, hydrogen peroxide (H₂O₂), sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), sulfosalicylic acid, formalin, potassium chloride (KCl), and other chemicals were from HiMedia Laboratories, Mumbai, India; mouse-specific lipocalin-2, IL-6, IL-1β, and TNF-α ELISA kits were procured from Ray Biotech, Norcross, United States. SYBR-Green master mix and cDNA synthesis kits were purchased from Bio-Rad, United States. HPLC with a diode array detector was from Agilent Technologies, Singapore. NucleoSpin DNA Stool kits were procured from Macherey-Nagel, Düren, Germany. Antibodies (occludin, zona occludin, claudin, GAPDH, Muc-4, and Alexa Fluor 488) from Cell Signaling Technology (Danvers, MA, United States). HRP (horseradish peroxidase)-conjugated secondary antibody and DNase treatment AM1906 were purchased from Thermo Fisher, Leica CTR6, and Leica Biosystems, Germany. A confocal microscope was purchased from Carl Zeiss LSM-880, Germany.

Glibenclamide (Glib) was procured from Ind-Swift Pvt. Ltd. (India). Alloxan and sodium carboxymethyl cellulose (CMC) were procured from SD Fine Chem Ltd. (SDFCL), Mumbai. Dibasic sodium phosphate, monobasic sodium phosphate, and diethyl ether (A.B. Enterprises, Mumbai); formalin, Folin-Ciocalteu reagent, thiobarbituric acid (TBA), glutathione (GSH), and dimethylsulfoxide (DMSO) (Hi-Media Lab Pvt. Ltd., Mumbai); ethylenediaminetetraacetic acid (EDTA) (Loba Chemie Pvt. Ltd; Mumbai); ethanol (Indian Chemical Co., Delhi); and aluminium trichloride (AlCl₃), sodium cyanide, sodium hydroxide (NaOH), sodium nitroprusside, sodium nitrite (NaNO₂), and sodium carbonate (Na₂CO₃) (Sisco Research Laboratory, Mumbai) were procured. Total cholesterol kit, HDL kit, and total protein kit were procured from Avecon Healthcare Pvt. Ltd., Saha (Ambala). The lactate dehydrogenase kit was procured from Reckon Diagnostics Pvt. Ltd., Gorwa, Vadodara. Creatine kinase isoenzyme (CKMB) was procured from Coral Clinical Systems, Industrial Estate, Verna, Goa.