Immunohistochemical staining was performed as previously described (22 (link)). The following primary antibodies were used: GFAP (SC-9065; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), COX-2 (12282; 1:200; Cell Signaling Technology, Inc.), PPAR-γ (2435; 1:200; Cell Signaling Technology, Inc.), NF-κB (6956; 1:200; Cell Signaling Technology, Inc.) and B-cell lymphoma extra-large (Bcl-xL; 2764; 1:200, Cell Signaling Technology, Inc.). Following incubation with primary antibodies at 4°C overnight, sections were washed in 0.1 M phosphate buffered saline (PBS) and incubated with anti-rabbit or anti-mouse horseradish peroxidase linked secondary antibodies (7074 or 7076; 1:2,000; Cell Signaling Technology, Inc.) for 1 h at 37°C. Following washing in PBS, sections were incubated with diaminobenzidine (PA110; Tiangen Biotech Co., Ltd., Beijing, China), and counterstaining with hemalum (AR0005; Boster Biological Technology, Pleasanton, CA, USA). Photomicrographic images were captured using a Leica DM4000B microscope (Leica Microsystems, Inc., Wetzlar, Germany) and Leica QWin plus version 4 software (Leica Microsystems, Inc.). For semi-quantitative analysis, the percentage of the positively stained area was measured at original magnification ×200 using Leica QWin plus software (Leica Microsystems, Inc.).
Qwin plus software
Manufactured by Leica
Sourced in United Kingdom, Germany
Leica QWin plus software is a comprehensive image analysis and processing solution designed for various microscopy applications. It provides a user-friendly interface for capturing, analyzing, and managing digital images acquired from Leica microscopes.
Automatically generated - may contain errors
Lab products found in correlation
Dm4000b microscope, by Leica (2 mentions)
Dfc490, by Leica (2 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Dfc300fx, by Leica (1 mentions)
Nissl staining solution, by Beyotime (1 mentions)
Fragel dna fragmentation detection kit, by Merck Group (1 mentions)
Application software, by Leica (1 mentions)
Dm2500, by Leica (1 mentions)
Ccd camera, by Leica (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Dm2500 light microscope, by Leica (1 mentions)
Dfc 490 digital, by Leica (1 mentions)
Diamond knives, by Electron Microscopy Sciences (1 mentions)
Cm100, by Hitachi (1 mentions)
Dmrb microscope, by Leica (1 mentions)
Reichert ultracut e ultramicrotome, by Reichert Technologies (1 mentions)
Paraffin, by Leica (1 mentions)
Automated tissue processor, by Leica (1 mentions)
13 protocols using qwin plus software
1
Immunohistochemical Staining of Neural Markers
2
Wound Healing Assay with Treatments
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HFB4 and BHK cells were cultured in six-well plates and then left for 24 h to form an adherent monolayer. This layer was then gently injured in a unidirectional way across the center of the well using a sterile pipette tip (1 mL). A gap distance equivalent to the external diameter of the pipette tip (0.5 mm) was produced and the elimination of the detached cells was completed by washing the wells with fresh medium twice. Different treatments (Vit E, Vit C, collagen, plain NE lacking Vit E, plain NE, Vit C-loaded NE, collagen-loaded NE, and a mixture of collagen and Vit C-loaded NEs (1:1)) were added at 50 µL/mL and the plates were incubated for 48 h. After washing the cells with phosphate buffer saline (PBS), they were fixed for 30 min with 3.7% paraformaldehyde. The cells were stained with crystal violet (1%) in 2% ethanol for 30 min. Micrographs of the stained monolayer were captured on a microscope and the gap distance was measured (when possible) by drawing lines through the edge of repopulating fibroblast cells and quantifying the cell-free distance remaining via the Leica Qwin-Plus software (Leica Microsystems, UK).
3
Histopathological Analysis of Liver Tissue
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The histopathological analysis was carried out using a light microscope (Leica DFC 300 FX) with a 40x microscopic objective. For histological and morphometric analysis, we obtained twenty randomly chosen images of histological slides that were prepared from the liver sections. These slides were scanned using the Leica Application software. Histological screening focused on the detection and grading of the following abnormalities: inflammatory reaction, sinusoidal congestion, degenerative hepatocyte lesions (hydropic and steatotic), sinusoidal congestion, hyperemia, bleeding focus, intralobular and portal granulomas, fibrosis, and necrosis. The changes in the experimental histopathological parameters present were semiquantified according to a scoring method as (0) absent, (1) mild, when present in approximately less than 33% in the total area of the tissue, (2) moderate, when present between approximately 33% and 66% in the total area of the tissue, and (3) intense, when present in approximately more than 66% in the total area of the tissue [19 (link), 20 ]. The counting of the number of inflammatory cells present in the hepatic lobes included the resident Kupffer cells. Slides were analyzed using the Leica Q-Win Plus software (Leica Microsystems, Inc., Buffalo Grove, Illinois, USA).
4
Histological Evaluation of Cellular Apoptosis
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Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, hematoxylin and eosin (H&E) and Nissl staining were performed according to the manufacturer's protocol of the following kits: FragEL™ DNA Fragmentation Detection kit (QIA33; Merck KGaA, Darmstadt, Germany), Hematoxylin-Eosin kit (AR1180; Wuhan Boster Biological Technology, Ltd., Wuhan, China), and Nissl staining solution (C0117; Beyotime Institute of Biotechnology, Haimen, China). Images were captured using a Leica DM4000B microscope and Leica QWin plus software (Leica Microsystems, Inc.).
5
Wound Healing Assay with Metformin and 5-ASA
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Caco-2 and HCT-116 cells were grown in 6 well plates and allowed to adhere for 24 h. Gently and slowly the monolayer was scratched in one direction with a new 1 ml pipette tip across the center of the well. The resulted gap distance therefore equals to the outer diameter of the end of the tip. The wells were then washed twice with medium to remove the detached cells. Cells were treated with metformin, 5-ASA or the combination of both substances for 48 h. Cells were washed twice with 1x PBS, then fix the cells with 3.7 % paraformaldehye for 30 min and they were stained with 1 % crystal violet in 2 % ethanol for 30 min. Photos were taken for the stained monolayer on a microscope. The gap distance was measured using the Leica Qwin-Plus software (Leica Microsystems, UK).
6
Histological Analysis of Liver Tissue
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After fixation, the tissue samples of the liver's right lobe and the hepatic fragment selftransplanted were processed according to routine histologic techniques and embedded in paraffin blocks. Paraffin blocks were sectioned using a microtome into 4-mm-thick sections, placed onto glass slides, and fixed for histologic staining. Staining was performed using hematoxylin-eosin and Masson trichrome stains.
All morphometric analyses were performed at the Multiuser Laboratory of the Research Center for Biological Sciences, Federal University of Ouro Preto. To count the number of inflammatory cells present in the hepatic lobes, measure the different liver areas, quantify the collagen fibers and the glycogen deposition, and determine the hepatic capsule thickness; we randomly obtained 20 images from histologic slides that were prepared from the liver sections. These slides were scanned using the Leica Application software and analyzed using the Leica Q-Win Plus software (Leica Microsystems, Inc, Buffalo Grove, IL) [11] .
All morphometric analyses were performed at the Multiuser Laboratory of the Research Center for Biological Sciences, Federal University of Ouro Preto. To count the number of inflammatory cells present in the hepatic lobes, measure the different liver areas, quantify the collagen fibers and the glycogen deposition, and determine the hepatic capsule thickness; we randomly obtained 20 images from histologic slides that were prepared from the liver sections. These slides were scanned using the Leica Application software and analyzed using the Leica Q-Win Plus software (Leica Microsystems, Inc, Buffalo Grove, IL) [11] .
7
Morphological Characterization of Fungal Isolates
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For morphological characterization, specimens were initially observed with a Leica 205C stereomicroscope (Leica Biosystems, Nussloch GmbH, Nussloch, Germany). The microscopical characteristics were analyzed by mounting asexual structures in clear lactoglycerol, and 50 measurements for each morphological parameter were carried out at a magnification of × 1,000 using a Leica DM2500 light microscope equipped with a Leica DFC 490 digital camera, coupled to a computer containing the Leica Qwin-Plus software. The morphological characteristics of the isolates were compared with the description of R. gossypii and R. pseudoglycines14 ,15 (link).
For examination on a scanning electron microscope (JOEL JSM-700 1F model), fragments of symptomatic dry leaves were fixed in 10 mm diameter copper stubs with double-sided carbon tape and coated with 25 mA gold, 1.10–2 mbar, for 2.5 min.
For examination on a scanning electron microscope (JOEL JSM-700 1F model), fragments of symptomatic dry leaves were fixed in 10 mm diameter copper stubs with double-sided carbon tape and coated with 25 mA gold, 1.10–2 mbar, for 2.5 min.
8
Tissue Microarray Immunohistochemistry Analysis
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Tissue microarrays were created and stained using standard protocols. A high-sensitivity diaminobenzidine chromogenic substrate system was used for colorimetric visualization. The density of positive staining was measured using a computerized image system and captured using a Leica-CCD camera connected to a Leica-DM-IRE2 microscope (Leica, GER). Under high-power magnification, photographs of representative fields were captured using the Leica Q Win Plus software (version 3). The IHC staining results were evaluated by two independent, experienced pathologists. Finally, according to pAMPK and a-SMA staining, their low and high expression groups were set for comparisons.
9
Macroscopic and Microscopic Fungal Characterization
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Macroscopical characters were observed from hyphal tip cultures grown on PDA, 2% Malt Extract Agar (MEA) and Sabouraud Dextrose Agar (SDA) during seven days at 25 ± 1 °C, while microscopical characteristics were studied with the fungus grown in MEA. Microscopical characteristics were analyzed by mounting reproductive structures in clear lactoglycerol, and 30 measurements for each morphological parameter were carried out at a magnification of × 1,000 using a Leica DM2500 light microscope equipped with a Leica DFC 490 digital camera, coupled to a computer containing the Leica Qwin-Plus software.
10
Morphological Characterization of Fungal Isolates
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For morphological characterization, specimens were initially observed with a Leica (Leica Biosystems, Nussloch GmbH, Nussloch, Germany) 205C stereomicroscope. The microscopical characteristics were analyzed by mounting asexual structures in clear lactoglycerol, and 50 measurements for each morphological parameter were carried out at a magni cation of × 1,000 using a Leica DM2500 light microscope equipped with a Leica DFC 490 digital camera, coupled to a computer containing the Leica Qwin-Plus software. The morphological characteristics of the isolates were compared with the description of R. gossypii and R. pseudoglycines 14, 15 (link) .
For examination on a scanning electron microscope (JOEL JSM-700 1F model), fragments of symptomatic dry leaves were xed in 10 mm diameter copper stubs with double-sided carbon tape and coated with 25 mA gold, 1.10-2 mbar, for 2.5 minutes.
For examination on a scanning electron microscope (JOEL JSM-700 1F model), fragments of symptomatic dry leaves were xed in 10 mm diameter copper stubs with double-sided carbon tape and coated with 25 mA gold, 1.10-2 mbar, for 2.5 minutes.
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