The kidney was resected from the rats in each group, incubated with the 4% paraformaldehyde (Sangon Biotech Co., Ltd., Shanghai, China) and embedded in the paraffin (Sangon Biotech Co., Ltd., Shanghai, China). The kidney tissues were cut into 4-μm thick sections and stained with hematoxylin (Nanjing Jiancheng Bioengineering Inst., Nanjing, China) and eosin (Biyotime Biotech Shanghai, China) and also sections were stained using a von Kossa staining kit (Beijing Leagene Biotech Co., Ltd., Beijing, China). The sections were observed by employing the polarized-light optical micro-photography (Mode: AX80, Olympus, Tokyo, Japan) to better confirm that the stained materials were the crystals. The formed crystals were evaluated using the professional image analysis software (NIH Image, version: 1.61, Scion Inc., Bethesda, MD, USA).
Hematoxylin
Manufactured by Nanjing Jiancheng
Sourced in China
Hematoxylin is a naturally occurring dye extracted from the heartwood of the Logwood tree (Haematoxylum campechianum). It is a commonly used stain in histology and microscopy, primarily for staining cell nuclei. Hematoxylin has the ability to bind to positively charged molecules, such as proteins, and produce a blue-purple color, allowing for the visualization of cellular structures under a microscope.
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Lab products found in correlation
Inverted microscope, by Olympus (4 mentions)
Diaminobenzidine, by Nanjing Jiancheng (4 mentions)
Paraformaldehyde (pfa), by Solarbio (4 mentions)
Trypsin, by Beyotime (3 mentions)
Maxvisiontm, by Maixin Group (2 mentions)
Eosin solution, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Paraffin, by Sangon (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Abcam (1 mentions)
Rm2145, by Leica camera (1 mentions)
Anti jak1, by Cell Signaling Technology (1 mentions)
Anti socs3, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti il 6, by Cell Signaling Technology (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Ab31013, by Abcam (1 mentions)
Ab39184, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
Ckx41 inverted light microscope, by Olympus (1 mentions)
Oil red o, by Abcam (1 mentions)
22 protocols using hematoxylin
1
Kidney Crystal Formation Analysis
2
Liver Tissue Analysis: Histology and Immunohistochemistry
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The liver samples were isolated and dehydrated using ethanol, cleared using xylene, embedded in paraffin, and sectioned at a thickness of 5 μm. The tissue sections were divided into 2 parts: 1 part for hematoxylin and eosin ( H&E) staining and 1 part for immunohistochemical assay. For H&E staining, the tissue sections were stained with hematoxylin (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and eosin (Beyotime Biotech. Shanghai, China) directly. For the immunohistochemical analysis, the tissue sections were de-paraffinized, rehydrated, and heated at 100°C for 10 min to retrieve antigens. Then, the tissue sections were treated using 0.3% hydrogen peroxide to block the endogenous peroxidase and were incubated using goat serum (Hyclone, Logan, UT, USA) for 30 min to block nonspecific binding. The tissue sections were then incubated with rabbit anti-rat CD34 monoclonal antibody (Cat. No. ab81289, 1: 3000, Abcam, Cambridge, MA, USA) and rabbit anti-rat vascular endothelial growth factor (VEGF) polyclonal antibody (Cat. No. ab231260, 1: 2000, Abcam) at 4°C overnight and then incubated using horseradish peroxidase-conjugated goat anti-rabbit IgG (Cat. No. ab6721, 1: 1000, Abcam) for 30 min at room temperature. Finally, the bound secondary antibody was visualized using a diaminobenzidine substrate kit (ZSGB Bio. Co. Ltd., Beijing, China).
3
Assessing Bone Regeneration via Histology
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The regenerations of newly-formed bones were assessed by H&E staining and Masson’s trichrome staining. Following micro-CT analysis, the specimens (injured skulls) were fixed with 4% paraformaldehyde and paraffin embedded. The histological sections (5-μm) were cut and prepared from the injured skull tissues using a freezing microtome (Model: RM 2145, Leica, Frankfurt, Germany). The sections were then incubated with xylene (SinoPharm Med. Co., Shanghai, China) for 15 min and washed with 100% ethanol for 10 min, 90% ethanol for 10 min, and 75% ethanol for 10 min, followed by gradient-ethanol dewaxing to water. Then, the sections were stained with hematoxylin (Jiancheng Bioengineering Institute, Nanjing, China) and eosin (Beyotime Biotech) to assess pathological changes in newly-formed bones. Moreover, sections were also stained with Masson (Leagene Biotech. Co., Beijing, China) to assess changes in collagen fibers in newly-formed bones, according to the manufacturer’s instructions. Images of H&E staining and Masson’s trichrome staining were captured through an optical microscope (Model: IX71, Olympus, Tokyo, Japan).
4
Histochemical Analysis of Collagen Proliferation
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After fixing with 10% formaldehyde solution, the samples were pretreated using standard protocols and stained with hematoxylin (Jiancheng, Nanjing, China) for 5 min, washed with distilled water for 1 min, and differentiated using 0.1% HCl for 1 min to wash back to blue. The samples were then stained using acid fuchsin (Yike, Guangzhou, China) solution for 8 min and washed with distilled water for 30 s, after which 1% phosphomolybdic acid solution (Yike, Guangzhou, China) was applied for 5 min, solid green (Yike, Guangzhou, China) applied for 3 min after drying, and tap water used to rinse the samples. The samples were dewaxed and sealed to observe the proliferation of collagen in the skin tissue.
5
Immunohistochemical Analysis of Lung Tissue
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Immunohistochemistry (IHC) analysis was carried out by using MaxVisionTM techniques (Maixin Bio, China) based on the manufacturer's instructions. Firstly, the lung tissue was fixed by 4% paraformaldehyde (Solarbio, Shanghai, China), dehydrated, and paraffin embedded. The 5 μm thick slides were obtained. The deparaffinization and hydration were performed, the slides were then incubated with 3% H2O2 (Sinopharm, China) for 10 min and 0.1% trypsin (Beyotime, China) for 20 min. The primary antibodies were incubated at 4°C overnight and then incubated with HRP-polymer-conjugated secondary antibodies at 37°C for 1 h. The slides were then stained by using the chromogenic reagent diaminobenzidine (DAB, Zhongshan, Beijing, China) for 3 min and counterstained with hematoxylin (Jiancheng, Nanjing, China). An inverted microscope (Olympus, Japan) was employed for image acquisition. The primary antibodies anti-IL-6, anti-JAK1, anti-STAT3, and anti-SOCS3 were purchased from Cell Signaling Technology (CST).
6
Hematoxylin and Eosin Staining Protocol
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The protocol of HE staining was similar to that used in a previous study [8 (link)]. Initially, rapid decalcification of the sections was performed in an oven with nitric acid at a low temperature and they were then embedded in paraffin following dehydration. When serial sections at the midsagittal area were obtained, they were prepared in 5-μm thickness by placing the sections in a parallel direction to the stab for staining 10 min using hematoxylin (Jiancheng, Nanjing), and washed with running water. The 1% hydrochloric acid-alcohol was used to decolorize the excess hematoxylin dye for 30 s. The samples were immersed in water for 15 min, stained with eosin solution (Beyotime, China) for 2 min, rinsed the excess dye with running water, dehydrated, and sealed with gum. Visualization and photography were performed under a microscope (OLYMPUS, Japan). The nucleus presented blue and the cytoplasm red.
7
Immunohistochemical Analysis of TGF-β, TIMP-3, and COL2A1
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The protocol of IHC was described by a previous study [8 (link)]. Following deparaffinization, the sections were incubated with 3% H2O2 (Sinopharm, China) for 10 min and 0.1% trypsin (Beyotime, China) for 20 min. Primary antibodies were subsequently incubated at 4°C overnight, using anti-TGF-β (ab31013, Abcam, USA), anti-TIMP-3 (ab39184, Abcam, USA), and anti-COL2A1 (ab34712, Abcam, USA). However, the treatment of negative control was conducted using PBS instead of the primary antibodies. The secondary antibodies used were HRP-conjugated and the section incubation was performed in a 37°C incubator for 30 min. Finally, the bound antibodies were stained with diaminobenzidine (DAB, Zhongshan, Beijing), and the sections were counterstained with hematoxylin (Jiancheng, Nanjing). Images were photographed by an inverted microscope (OLYMPUS, Japan).
8
Histological analysis of rat gastric tissue
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The gastric tissues of the rats were fixed in 4% formalin at 25˚C for 24 h and underwent routine ethanol dehydration and paraffin embedding. The embedded tissues were cut into 5-µm slices using a Leica Biosystems RM2245 wheel slicer (Leica Microsystems GmbH). The slices were baked in an oven at 60˚C for 2 h and were then dewaxed and rehydrated. Subsequently, the slices were stained with hematoxylin (Nanjing Jiancheng Bioengineering Institute) at 25˚C for 3 min and eosin (Nanjing Jiancheng Bioengineering Institute) at 25˚C for 30 sec. The stained slices were dehydrated with ethanol, cleared with xylene, mounted on glass slides with neutral balsam and covered with coverslips. The rat gastric tissues were imaged using the Olympus CKX-41 inverted light microscope (Olympus Corporation) at x100 magnification. The atrophy and inflammation of gastric glands were scored according to the visual analogue scale of the new Sydney system (16 (link),17 (link)). The pathology scoring was carried out independently by two examiners, and the mean value was finally taken.
9
Quantification of Ox-LDL-Induced Lipid Accumulation
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After stimulated with LPS for 24 h, the culture medium was changed for fresh medium containing 50 μg·ml−1 of ox-LDL (Peking Union Bio, China). To analyze the lipid accumulation, the cells after different treatments were fixed by 4% paraformaldehyde and dehydrated in 60% isopropanol for 2 min. Then, the cells were stained with filtered 0.3% Oil Red O (Abcam, USA) for 30 min and hematoxylin (Jiancheng Biotech, China) for 1 min. Finally, wash of PBS buffer, the cells washed by PBS buffer and were examined by light microscopy to observe the lipid accumulation in macrophages.
10
Histopathological Analysis of Ischemic Brain
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The mice were sacrificed at 2 days after I/R. The brain tissues of the mice were removed rapidly and sliced into 8-µm-thick coronal sections on a freezing microtome. The brain sections were stained with hematoxylin (Jiancheng Biotech) for 5 min and differentiated with 1% hydrochloric acid alcohol for 5 s. The sections were then placed in ammonia water for 10 s, stained with eosin for 3 min, dehydrated, cleared with alcohol and xylene, and finally sealed with neutral balsam. Histopathological changes in the ischemic brains were observed under a microscope.
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