Icc50

The DeadEnd™ Colorimetric TUNEL System was used to detect apoptotic cells in situ in cultured cells, according manufacturer’s instructions. The systems end-label the fragmented DNA of apoptotic nuclei using a modified Tunel method. Briefly, U373 were seeded on Lab-Tek^® Chamber Slides (10⁴ cell/well) and treated with BCP at doses of 20 and 30 μg/mL for 24 h. Then, cells were fixed in 4% paraformaldehyde solution in PBS for 25 min at room temperature. After permeabilization with 0.2% of Triton–X100 solution in PBS, for 5 min, and rinsing with PBS, cells were incubated with a mix containing biotinylated nucleotide and Terminal Deoxynucleotidyl Transferase, Recombinant, (rTdT) enzyme for 60 min at 37 °C. After rinsing, slides were incubated with a streptavidin HRP for 30 min at room temperature. Following the addition of the chromogen diaminobenzidine (DAB), apoptotic nuclei are stained dark brown and visualized with a light microscope (20× objective, Leica ICC50).
A total of 20 representative images were investigated for each sample and the number of apoptotic cell was determined by counting the total number of nuclei per image area and the TUNEL-positive nuclei. The results were expressed as % of apoptotic cells.

The tissue sections were canvassed with a microscope (Leica DM750) with an attached Leica ICC50 digital camera. Photomicrographs of the tissue sections were considered at diverse magnifications.

Liver samples were fixed in a 10% buffered neutral formalin solution and were transferred into an automatic processor (Leica TP 1020, Buffalo Grove, IL, USA) where they were dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Samples were sectioned at 5 μm thickness by using a rotary microtome (Medite M380, Burgdorf, Germany). The sections were stained with hematoxylin and eosin (H&E) and then were examined using a Leica light microscope (Leica DM750, Wetzlar, Germany), and provided with a camera (Leica ICC50, Wetzlar, Germany). For each liver specimen, tissue changes were examined in 10 randomly selected areas. The microscopic appearance of the liver tissues was examined for fatty vacuolation, hepatocyte necrosis, hepatocyte ballooning, massive micro and macrovesicular, intracellular lipid droplets in hepatocytes and inflammatory cell infiltration. The number of apoptotic cells, necrotic cells and lipid droplets were measured using the particle sizing function provided by the ImageJ software version 1.53 (Rasband, ImageJ, National Institutes of Health, Bethesda, MD, USA). Counting cells was assessed in triplicate using the following equation: $$\mathrm{Counting} \mathrm{cells} (\%)=\frac{\left(\mathrm{count} \mathrm{target} \mathrm{cells}\right)}{\left(\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{hepatic} \mathrm{cells}\right)}\times 100$$

A cytoblock was performed with dissociated R2J-GS cells fixed in 4% formol for 30 min and prepared with Shandon™ Cytoblock™ Reagent (Fisher scientific). The brains of mice were also fixed in 4% formol, and the cytoblocks were embedded in paraffin. HES and IHC analyses were performed from 3 µm paraffin sections using the Histostain^® Plus Kit and the Bond Polymer Refine Detection Kit (Leica DS9800) (Leica Biosystems, Newcastle, UK).
Anti-Olig2 (HPA003254) is a Sigma Life Science product. Anti-GFAP (ab33922), anti-CD44 (ab51037), anti-CD34 (ab110643) anti-nestin (ab93666), and anti-NCAM1 (ab75813) were obtained from Abcam (Cambridge, UK). Slides were incubated for 1 h with diluted antibodies (1/2000 for Olig2, 1/1000 for GFAP, 1/100 for CD44, 1/6400 for CD34, 1/200 for Nestin, 1/2000 for CD56) at room temperature before peroxidase revelation according to the Leica protocol. Pictures were captured using a Leica ICC50 camera connected to a Leica DM250 microscope (objective x20).

The G. butleri strain GbKAU was isolated and purified from the organic compost at Kerala Agricultural University, India, following the standard protocol [21 (link)]. Cultural characteristics such as colony appearance and mycelial texture were observed by growing the culture on potato dextrose agar medium (PDA) and incubating at 28 °C for 3–7 days. Pure isolates were obtained by picking fungal tips. Microscopic analysis was carried out by staining with lactophenol cotton blue stain. Observations were made using a camera-supported microscope (Leica ICC50, Germany) at 40× magnification. The type of mycelium, characteristics of sporangia, and arrangement of sporangiophore were recorded. The identity was cross-verified in the studies conducted at the National Center of Fungal Taxonomy, New Delhi.