Rats were anesthetised with sodium pentobarbital (100 mg/kg, intra-peritoneal) and perfused trans-cardially with paraformaldehyde (4%) in phosphate buffer (0.1 M). The brains were removed and placed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer overnight. The brains were dehydrated and embedded in wax. Coronal sections of 5 mm were harvested at approximately -3.6 mm from the bregma relative to adult brain and then stained with 0.5% cresyl violet. The microphotograph was taken at 20× using a BX-60 light microscope (Olympus, Southall, UK) attached with a digital camera (Zeiss, Gottingen, Germany). The following morphologic criteria were used to analyse apoptotic or necrotic cell death in the cerebral cortex [44 (link)]. Necrotic or apoptotic cells were identified by dark-stained, shrunken nuclei that were spherically shaped with loss of nuclear membrane integrity. The total number of healthy cells that appeared in the cortex in cresyl violet staining slices were counted in a blinded manner and their mean value used for data analysis.
Digital camera
Manufactured by Zeiss
Sourced in Germany, United States
The Digital Camera is a compact, high-performance imaging device designed for laboratory applications. It features a state-of-the-art image sensor and advanced image processing capabilities, delivering precise and detailed visual data for scientific analysis.
Automatically generated - may contain errors
Lab products found in correlation
Inverted microscope, by Zeiss (4 mentions)
Apotome microscope, by Zeiss (2 mentions)
Dapi mounting medium, by Vector Laboratories (2 mentions)
Matrigel, by BD (2 mentions)
Apotome, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bx60 light microscope, by Olympus (1 mentions)
Inverted fluorescent microscope, by Zeiss (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Optical microscope, by Zeiss (1 mentions)
Apreo scanning electron microscope sem, by Thermo Fisher Scientific (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Jenamed 2, by Zeiss (1 mentions)
Compound microscope, by Zeiss (1 mentions)
Axioskop microscope, by Zeiss (1 mentions)
Axiocam mrc camera, by Zeiss (1 mentions)
Digoxigenin rna labeling kit, by Roche (1 mentions)
Discovery v12, by Zeiss (1 mentions)
68 protocols using digital camera
1
Quantifying Neurodegeneration in Rat Cortex
2
Quantifying Adipocyte Morphology in vWAT
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Five-micrometer-thick sections of paraffin-embedded vWAT were stained with Mayer's Hematoxylin and Eosin (H&E). Images of the staining were captured at X2000 magnification using a Zeiss Axioscope microscope equipped with a digital camera (Carl Zeiss, Thornwood, NY). Adipocyte area and average diameter were determined using Adiposoft, a software program that utilizes the NIH's open-share Fiji program (http://sw.wikkii.com/wiki/Adiposoft ). Macros were developed to determine which cells to include in the size calculations (they had to be above a certain level of circularity and size to exclude pre-adipocytes or other cell types), and the area and Feret's diameter were calculated for each adipocyte assigned to each animal. For each mouse, 25–35 adipocytes from three separate sections were analyzed to determine the mean adipocyte volume.
3
Transwell Assay for Evaluating MEC Migration
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HMEC-1 cells were cultured in Transwell units with polycarbonate filter and 8 μm pores (Costar) in 24-well plates for 2 days at 37 °C to confluence. MECs were starved overnight and labeled with CellTracker Green CMFDA Dye. An aliquot (1 × 105/well) of fluorescent MECs in serum-free medium was added in the top of the Transwell chamber, whereas the conditioned medium was used as a chemoattractant in the lower compartment. The wells containing the serum-free medium in the lower compartment were used to estimate the background migration. MECs were migrating across the endothelial monolayer for 6 h in a humidified incubator (temperature of 37 °C and 5% CO2 atmosphere). At the end of the assay, non-migrated cells were wiped out from the inner surface of the membrane with a cotton swab. The assay was documented at 200 × magnification with a fluorescent inverted microscope and a digital camera (Zeiss). The migrated cells (five randomly chosen fields of each well) were counted using the ImageJ software.
4
Immune Synapse Imaging and Granzyme B Polarization Assay
5
Immunohistochemical Analysis of Retinal Inflammation
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Eyes were enucleated on day 7, post hypertonic saline injection and placed in 4% PFA for 12 h at 4°C. The retinas were then transferred to 30% sucrose for 12 h followed by embedding the retinas in OCT on dry ice. Cryosections were cut at −30°C and stored at −20°C until used for immunohistochemistry. Retina sections were rinsed with Tris-buffered saline (TBS) at room temperature followed by permeabilization using 0.5% Triton-X-100 in TBS. The tissues were blocked with 5% BSA for 1 h followed by incubation with primary antibodies (anti-IL-1β, anti-IL-6, or anti-phospho-STAT3) for 16 h at 4°C. After washing, the tissues were incubated with secondary antibody (Alexa Fluor 488 or Rhodamine) for 1 h at room temperature. The details of antibodies dilution and commercial sources are provided in Table 2 . The sections were observed under a bright-field microscope equipped with epifluorescence, and digitized images were captured by a digital camera (Zeiss) as described earlier (Husain et al., 2012a (link); Abdul et al., 2013 (link); Husain et al., 2014 (link); Husain et al., 2017 (link)). We have used four animals in each group of treatment and a total of 12 animals were used for Immunohistochemistry.
6
Optic Nerve Immunohistochemistry After Injury
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The optic nerves were collected on day 7 after injury and placed in 4% paraformaldehyde for 12 to 16 hours at 4°C. The optic nerves were then transferred to 30% sucrose for 24 hours followed by embedding the optic nerves in optimal cutting temperature (OCT) on dry ice. Cryosections were cut at –30°C and stored at –20°C until used for immunohistochemistry. Tissue sections were rinsed with Tris-buffered saline (TBS) three times at room temperature followed by permeabilization using 0.5% Triton-X-100 in TBS. The tissues were blocked with 5% BSA in TBS for 1 hour and then rinsed again with TBS followed by incubation with primary antibodies (anti-GFAP, dilution 1:500) for 16 hours at 4°C. After washing, the tissues were incubated with secondary antibody (Alexa Fluor 488, dilution 1:500) for 1 hour at room temperature. The sections were observed under a bright-field microscope equipped with epifluorescence, and digitized images were captured by a digital camera (Carl Zeiss Meditec, Jena, Germany) as described elsewhere.8 (link),16 (link),21 (link)
7
Histochemical Analysis of Hepatic Glycogen
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Small pieces of the left lobes of liver were fixed in methacarn for 3 h, stocked in 70% alcohol and then routinely processed for paraffin-embedded. The blocks were cut into 4-μm-thick, obtaining semi-serial sections that were disposed on light microscopy slides. Detection of glycogen was obtained by the immersion of the slides in 1% periodic acid solution for 10 min and, after subsequent washes in water, sections were submitted to Schiff-reactive for 30 min. The sections were counter-stained with Harris’ hematoxylin. Slides were analyzed by common light microscopy connected to digital camera (Zeiss) and photomicrographs were obtained under 20x objective.
For glycogen analysis in hepatic samples, two different zones were considered as follows: zone I, located around the portal tracts and zone III, encircle the central veins. According to the intensity of zones staining, the amount of glycogen was classified as light, moderate or intense.
For glycogen analysis in hepatic samples, two different zones were considered as follows: zone I, located around the portal tracts and zone III, encircle the central veins. According to the intensity of zones staining, the amount of glycogen was classified as light, moderate or intense.
8
Immunohistochemical Analysis of Tumor Markers
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Paraffin‐embedded sections of tumors were stained with H&E using standard methods. Immunohistochemical staining was carried out using Abs from Cell Signaling Technology or Abcam (Cambridge, UK). Staining was undertaken using frozen sections of tissue embedded at an optimal temperature. The images were captured with a Carl Zeiss digital camera (Stuttgart, Germany) attached to a light microscope. The number of Ki‐67‐ and cleaved caspase‐3‐positive cells was quantified.
9
Immunohistochemical Analysis of IL6R
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The paraffin sections were dewaxed, hydrated, and incubated with 3% H2O2 for 10 mins to inactivate the endogenous peroxidase activity. Subsequently, the tissue slides were washed with water and PBS for 3 times. Goat serum (10%) was used to block the nonspecific binding of the primary antibody to the tissue slides, which were subsequently incubated for 10 mins at room temperature, followed by an additional incubation with antibodies against IL6R (item no. 373708; Santa Cruz Biotech, Inc., TX, USA) at 37°C for 2 hrs. The tissue slides were washed with PBS for 3 times, followed by incubation with the secondary HRP-conjugated antibody (Santa Cruz Biotech, Inc., TX, USA) at 37°C for 30 mins. The tissue slides were stained with DAB substrate chromogen solution (Histolab Products AB, Goteborg, Sweden), washed with water, counterstained with hematoxylin, dehydrated, vitrified and laminated. A microscope (Carl Zeiss, Jena, Germany) was applied for the observation of the tissue slides and the images were acquired using a digital camera (Carl Zeiss, Jena, Germany).
10
Morphological Characterization of Transfersomes
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The transfersomes were detected, and then photos were captured under an optical microscope fitted with a digital camera (Carl Zeiss, Germany) at 40X magnification. This test was performed before particle size reduction by ultrasonication and extrusion.
Furthermore, the surface topography of the selected F15 and the blank formula was examined by Thermo Scientific Apreo Scanning Electron Microscope SEM (Czech Republic).
The lyophilized samples were placed on an aluminum holder using carbon tape, followed by a gold coating. Afterward, the samples were subjected to SEM imaging.
Furthermore, the surface topography of the selected F15 and the blank formula was examined by Thermo Scientific Apreo Scanning Electron Microscope SEM (Czech Republic).
The lyophilized samples were placed on an aluminum holder using carbon tape, followed by a gold coating. Afterward, the samples were subjected to SEM imaging.
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