Sodium azide

2′,7′ − Dichlorofluorescin Diacetate (DCFDA), Vanadium chloride (III), Prednisolone (purity 99%), cetyltrimethylammonium bromide (CTAB), Xylenol orange and Trolox were purchased from Sigma − Aldrich (Burlington, USA). Bovine serum (BSA), o − dianisidine dihydrochloride (ODD), methionine, reduced Glutathione (GSH), oxidised Glutathione, nicotinamide adenine dinucleotide (NADH), hydroxylamine hydrochloride, 2,4 − dinitrophenylhydrazine and riboflavin were purchased from Sisco Research Laboratory (Mumbai, India). Sodium azide, 5, 5 − Dithiobis − 2 − Nitrobenzoic acid (DTNB) and 1 − chloro − 2,4 − di nitro benzene (CDNB) were purchased from Himedia (Maharashtra, India).

The purchased chemicals and kits from respective company used were Propidium Iodide (PI) (Calbiochem, #537059), Ribonuclease A (RNase A, Biotech, #9001-99-4.), ethanol (Sigma Aldrich, #1009832500), PBS (Sigma Aldrich, #P4417), fetal bovine serum (FBS) (Gibco, USA), antibiotic (Himedia, #A018), Iscove's Modified Dulbecco's Medium (IMDM) (Sigma Aldrich, #I7633), N (Calbiochem, #565851), Human S (Sigma Aldrich, #57901), BrdU colorimetric cell proliferation kit (Calbiochem, #JA1599), 5(6)-carboxyfluorescein diacetate N succinimidyl ester (CFDA-SE) (Sigma Aldrich, #21888), PrestoBlue (Invitrogen, #A13261), Paraformaldehyde (PFA) (Sigma Aldrich, #P6148), Ki-67 mouse monoclonal IgG antibody (SantaCruz, #23900), Goat Anti-mouse IgG FITC Conjugated (Sigma Aldrich, #F5387), Bovine Serum Albumin (BSA) (AMRESCO, #0332), sodium azide (HiMedia, #GRM1038), saponin (Calbiochem, #558255), StemPro-34 nutrient supplement (Gibco, #10641), CyQuant cell proliferation assay kit (Invitrogen, #35011), Anti-CD117 antibody (c-Kit-PE, Millipore, #10482), Anti-Phosho c-Kit (pY568 and pY570) antibody (Abcam, #ab5616), Rabbit monoclonal Anti-SHP-1/2 (Millipore, #04742), Goat Anti-rabbit IgG-R-PE (Invitrogen, #PZ771MP), Mouse Anti-HePTP (PTPN7) IgG2b_κ (Millipore, #04278), Anti-mouse IgG2b FITC (Sigma Aldrich, #SAB3701184).

Benzylaminehydrochloride (BAHC), bovine serum albumin (BSA), butylated hydroxy toluene (BHT), 1-chloro-2, 4-dinitrobenzene (CDNB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), epinephrine, oxidized glutathione (GSSG), reduced glutathione (GSH), hydrogen peroxide (H₂O₂), nicotinamide adenine dinucleotide phosphate (NADPH), o-phosphoric acid (OPA), thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 1-amino-2-naphthol,4-sulfonic acid (ANSA), 2,4-dinitrophenyl hydrazine (DNPH), ethylenediaminetetraacetic acid (EDTA), and sulfosalicylic acid (SSA) were purchased from Merck Limited (Mumbai, India). Guanidine hydrochloride and sodium azide were obtained from Hi-Media Labs (Mumbai, India). STZ and HP were obtained from Sigma Aldrich (St. Louis, USA).

All the media were purchased from Himedia, India. Giemsa's azur eosin methylene blue solution and Hematoxylin and eosin stains for microscopy were obtained from Merck KGaA Frankfurter Str., Germany. A phosphate buffer solution (PBS), sodium bicarbonate, sodium azide, RNase A, NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) (Cat# RM 578, RM 2577) were procured from Himedia chemicals, India. Antibodies against TGF-β, TNF-α, β-actin, AP-linked anti-rabbit, AP-linked anti-mouse secondary antibodies were purchased from the Cell signaling Technology Cell Signaling Technology, Inc (Danvers, MA, USA). Pre stain molecular weight protein markers were purchased from Bangalore Genei (India) and the remaining chemicals were purchased in an analytical grade of the highest purity (India). All solutions were prepared with commercial reagents of at least pro-analysis quality and with sterilized 18 MΩ milliQ water. When necessary, the specific origins of reagents are listed in the text.

The O₂⁻ content was estimated spectrophotometrically by following the method given by Wu et al. [60 (link)]. Briefly, one gram of fresh plant sample was grounded in phosphate buffer (6 mL of 65 mM, pH 7.8) containing 2% polyvinylpyrrolidone, followed by centrifugation (at 5000× g for 15 min at 4 °C). The mixture was incubated at 25 °C for 30 min. After that, 0.5 mL of supernatant was mixed with 0.5 mL of phosphate buffer and 0.1 mL of hydroxylamine hydrochloride (10 mM, Sigma Alrdrich, Mumbai, India) and incubated again. Incubated mixture was than mixed with 3-amino benzene sulphonic acid and 1-naphthylamine (1 mL of 58 and 7 mM respectively, CDH, New Delhi) and kept at 25 °C for 20 min. The absorbance was recorded at 530 nm and the amount of O₂⁻ was calculated against the sodium nitrite (Himedia, Mumbai, India) as standard which was expressed as μmol g⁻¹ FW. The in-vivo visualisation of O₂⁻ was done by using the method mentioned by Frahry and Schopfer [61 (link)]. The leaves of B. juncea were first incubated in nitro blue tetrazolium (NBT, Himedia, Mumbai, India) prepared in potassium phosphate buffer with pH 6.4 and sodium azide (10 mM), followed by a process of decolouration (by immersing them in boiling ethanol), and photographed.