For immunohistochemistry staining, the liver tissues were first fixed in a solution with 4% paraformaldehyde; they were then embedded in paraffin. Sections of 4 μm thickness were then cut from these paraffin-embedded tissues. The antigen repair solution with citric acid (pH 6.0, Service Bio, China) was employed to enhance antigen retrieval. After this step, the slides were treated with a 10% rabbit serum (Service Bio) to block non-specific binding. The slides were placed in primary antibody (Table 2 ) and stored at 4°C overnight. Following this, the slices were subjected to DAB (3,3′-diaminobenzidine) treatment. Subsequently, the slides were dehydrated using alcohol and subjected to hematoxylin (Service Bio, China) staining. The stained slides were examined under a microscope (Nikon E-100 Tokyo, Japan). Images were captured using the Case Viewer program (3DHISTECH, Hungary Ltd.).
Caseviewer program
Manufactured by 3DHISTECH
Sourced in Hungary
CaseViewer is a program developed by 3DHISTECH for viewing and analyzing digital pathology slides. The software allows users to open, navigate, and examine high-resolution digital images of tissue samples. CaseViewer provides basic functionalities for image manipulation and annotation.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Citric acid ph 6, by Wuhan Servicebio Technology (1 mentions)
Digital slide scanner, by 3DHISTECH (1 mentions)
Pannoramic 250 flash 2 digital slide scanner, by 3DHISTECH (1 mentions)
Panoramic scan, by 3DHISTECH (1 mentions)
Pannoramic 250 flash 3 slide scanner, by 3DHISTECH (1 mentions)
6 protocols using caseviewer program
1
Immunohistochemistry Staining of Liver Tissue
2
Quantitative Immunohistochemical Analysis
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Representative images of the immunohistochemical stainings were obtained using a digital slide scanner (3DHISTECH, Sysmex, Budapest, Hungary). Subsequently, separate image sections of the tumor and tumor-surrounding normal colorectal tissue were created at a 10-fold magnification using the Case Viewer program (3DHISTECH, Sysmex, Budapest, Hungary). The semi-quantitative analysis of the images was carried out using ImageJ (NIH) as previously described [22 (link)]. In brief, the images were converted into 8-bit greyscale images and inverted. Each pixel of the image was assigned an intensity value between 0 and 255. To exclude non-specific low background pixels and intensities from the analysis, a lower threshold of 50 was set. Finally, the mean intensity values for all pixels of an image were generated by measurements in ImageJ.
3
Quantitative Nasal Mucosal Analysis
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Prior to hematoxylin and eosin staining, all sections were deparaffinized with xylene and then rehydrated in a graded ethanol/distilled water series. The slides were scanned using the Pannoramic MIDI digital slide scanner (Pannoramic software 2.0; 3DHISTECH Ltd., Budapest, Hungary). Five coronal sections that were similar to the sinus cavity were chosen for evaluation to minimize processing errors and confirm the presence of mucosal lesions. Ten areas of nasal mucosal sections in the maxillary sinus or lateral wall of the nasal cavity were chosen randomly for the evaluation under high-power fields (×400) and measured by 2 examiners who were blinded regarding the groups to which the animals had been assigned. Polypoid lesions were defined as distinct mucosal elevations with inflammatory cell infiltration around the microcavities. The thickness of edematous mucosa was measured as the distance between the apices of epithelial cells and the upper border of the subepithelial glands zone. For the assessment of mucosal thickness, at least 3 measurements at random points were made in appropriate areas of each high-power field; the mean values from 4 different high-power fields were recorded for comparison. Thickness was measured using the CaseViewer program (v: 1.15.3; 3DHISTECH Ltd.).
4
Uterine Adenomyosis Analysis in Mice
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The uteri were fixed in 4% paraformaldehyde at room temperature for 15–20 h, dehydrated and paraffin-embedded. HSD17B1TG mice do not cycle and WT uterus samples were collected at random cycle phases. Serial sections of 16 WT and 6 HSD17B1TG mice at the age of 5.5 months were cut at 5 μm thickness and stained with hematoxylin and eosin. One uterine horn was cut into 4–5 pieces. The uterus piece second closest to the ovarian end was cut through and used for the analysis for each mouse. On average, 133 ± 9 and 220 ± 16 sections for WT and HSD17B1TG mice were analyzed, respectively. For the time point of 12 months, one slide per uterus, containing an average of 48 ± 5 sections for WT and 51 ± 6 for HSD17B1TG mice, was analyzed from the uterus piece second closest to the ovary. Endometrial glands partially or fully infiltrated into myometrium were considered as a sign for adenomyotic changes in all time points. All slides were scanned with Pannoramic 250 Flash II digital slide scanner (3DHISTECH, Budapest, Hungary). The gland infiltration depth was measured using the Caseviewer program (3DHISTECH, Budapest, Hungary).
5
Histomorphometric Analysis of Pancreatic and Adipose Tissues
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The tissues were fixed in 10% neutral formalin, embedded in paraffin, sectioned (thickness: 5 µm), and stained with H&E. Images of the tissues were captured using Panoramic SCAN (3DHISTECH, Budapest, Hungary), and the pancreatic islet area and the number of beta cells were measured using the CaseViewer program (3DHISTECH, version 2.2). The iWAT, eWAT, and BAT adipocyte areas were measured using the adipocyte tool plugin in ImageJ software 1.52a (NIH, Bethesda, MD, USA).
6
Histological Analysis of Emb Knockout Mice
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Male and female WT and Emb-/- mice were examined at the age of 2, 4, and 6 months. Three to four mice were included in each independent study group. Additionally, one male and one female Emb-/- mouse were analyzed at the age of 1 year. The mice and the specific organs: heart, lung, liver, spleen, kidney, epididymis, testis, and ovary, were weighted and prepared for histological analysis. Also, the samples of skin, small intestine, and adrenal glands were prepared for histological analysis. Lungs were additionally analyzed from 11 WT and 14 Emb-/- embryos from six E17.5 Emb+/- breedings and 10 WT and 7 Emb-/- pups from four P0 Emb+/- breedings. Placentas were analyzed at E17.5. Sections (4 μm) were cut and immobilized to adhesion slides as mentioned earlier. The sections were deparaffinized, rehydrated, and stained with conventional hematoxylin and eosin (H&E), imaged with Pannoramic 250 Flash III slide scanner (3D Histech), and analyzed with the CaseViewer program (3D Histech). In the case of H&E-stained histological lung sections from E17.5 embryos and P0 pups, three images per organ section at 20x magnification were selected with the CaseViewer program. The relative area of airways in the lung section images was analyzed with ImageJ/Fiji.49 (link)
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