Naphthol as d chloroacetate specific esterase kit

After anesthetization, the mice were transcardially perfused with ice-cold 4 % formalin. Then, the brains were removed and fixed in 4 % formalin for 48 h. The formalin-fixed tissues were embedded in paraffin and then sliced into 4μm sections. Infiltrating neutrophils were stained using a Naphthol AS-D Chloroacetate Specific Esterase Kit (Sigma, St. Louis, MO, USA). We selected more than four fields of view at a primary magnification of ×200 in the cortex or hippocampus of every brain section. Cells were counted under a Nikon E100 microscope, and the data are presented as the means ± SEM.

Esterase activity of cells of granulocytic lineage was determined by a Naphthol AS-D Chloroacetate (Specific Esterase) Kit (Sigma), according to the manufacturer's instructions. Briefly, liver cryostat sections were fixed in citrate-acetone-formaldehyde solution for 30 seconds prior to incubation at 37°C in naphthol AS-D chloroacetate solution. Slides were then rinsed and mounted for analysis under the microscope.

Collected samples were fixed in 10% neutral buffered formalin (Bamed, Ceske Budejovice, Czech Republic). Subsequently, they were histologically processed and stained with hematoxylin and eosin (both from Merck, Kenilworth, NJ, USA), according to Pejchal et al. [28 (link)]. The histopathological analysis was performed using a BX-51 microscope (Olympus, Tokyo, Japan) and a semiquantitative scale developer by Shelby et al. [20 (link)].
Neutrophil granulocytes were detected using Naphthol AS-D Chloroacetate (specific esterase) Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Naphthol AS-D Chloroacetate positive cells were measured in 6 randomly selected microscopic fields in the mucosal and submucosal compartments at 400fold original magnification on a BX-51 microscope.

The materials are as follows: recombinant TNF-α (PeproTech, NJ, USA); ABT-199, SP600125, and lenalidomide (MedChemExpress, NJ, USA); TNF-α, myeloperoxidase (MPO), interleukin-1 β (IL-1β), IL-6, IL-8, and IL-18 enzyme linked immunosorbent assay (ELISA) kits (BD Biosciences, CA, USA); rabbit anti-mouse FoxO3a antibody, rabbit anti-mouse Ly6G antibody, rabbit anti-mouse JNK antibody, rabbit anti-mouse Bcl-2 antibody, rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, horseradish peroxidase- (HRP-) labeled goat anti-rabbit secondary antibody (Abcam, Cambridge, UK); HRP-labeled anti-mouse secondary antibody, bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology, Shanghai, China); Naphthol AS-D Chloroacetate (Specific Esterase) Kit (Sigma, NY, USA); Diaminobenzidine DAB Kit (ZLI9019; ZSGB-BIO); Dulbecco's modified Eagle's medium (DMEM) medium, trypsin, and fetal bovine serum (FBS) (Gibco, NY, USA); FoxO3a small interfering ribonucleic acid (siRNA) (Guangzhou RiboBio Co., Ltd., Guangzhou, China); and Cell counting kit-8 (CCK-8) and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) Apoptosis Assay Kit (Jiangsu KeyGEN BioTECH Corp., Ltd., Jiangsu, China).