The tumor tissue sections (4μm) were deparaffinized using xylene (Sinopharm Chemical Reagent Co., Ltd, #10023418, China), followed by rehydration with graded alcohol (Sinopharm Chemical Reagent Co., Ltd, #10009218, China). To stain the nuclei, the sections were subjected to hematoxylin staining at room temperature for 30 min, using hematoxylin (Solarbio, #G1121, China). After staining, the sections were washed with PBS to remove any excess hematoxylin and prevent over-staining. Next, 0.1% ammonia water (Solarbio, #G1823, China) was applied to the sections to change the color of the hematoxylin-stained nuclei from reddish to a distinct blue-purple hue. To prepare the sections for cytoplasm staining, they were rinsed with 75% alcohol at room temperature for 2 min. Cytoplasm staining was performed using eosin (Solarbio, #G1121, China) at room temperature for 1 h. After eosin staining, the sections were directly rinsed with graded alcohol to remove excess dye and prepare them for further analysis. Finally, the sections were treated with xylene, which acted as an anhydrous alcohol, before being mounted on slides. The sections were examined using a light microscope (Leica), and the images were analyzed with Image-Pro Plus software (version 6.0).
Hematoxylin
Manufactured by Solarbio
Sourced in China, United States, Japan
Hematoxylin is a naturally occurring dye extracted from the heartwood of the Logwood tree. It is commonly used as a staining agent in histology and cytology for the visualization of cellular structures, particularly nuclei, during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Solarbio (16 mentions)
Optical microscope, by Olympus (12 mentions)
Goat serum, by Solarbio (10 mentions)
Paraformaldehyde (pfa), by Solarbio (10 mentions)
In situ cell death detection kit, by Roche (7 mentions)
Bovine serum albumin (bsa), by Sangon (7 mentions)
3 3 diaminobenzidine (dab), by ZSGB-BIO (5 mentions)
Xylene, by Sinopharm (5 mentions)
Oil red o, by Solarbio (5 mentions)
Normal goat serum (ngs), by Solarbio (5 mentions)
Paraformaldehyde (pfa), by Sinopharm (5 mentions)
Bx53 microscope, by Olympus (4 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Image pro plus software, by Media Cybernetics (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Xylene, by Solarbio (3 mentions)
Ab205718, by Abcam (3 mentions)
H8070, by Solarbio (3 mentions)
Oil red o, by Merck Group (3 mentions)
314 protocols using hematoxylin
1
Hematoxylin-Eosin Staining of Tumor Tissue
2
Immunohistochemical Analysis of Tumor Biomarkers
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Sections of paraffin-embedded tumor tissue were deparaffinized and subjected to graded ethanol. Then, they were incubated with antibodies against p53, HMGCS1, HMGCR, and FDFT1 proteins, the same as those used in western blotting. Based on the manufacturer’s instructions, color development was performed using DAB chromogenic kit (Dako, cat#K5007). Hematoxylin (Solarbio, cat#H8070-5g) was used to counterstain for 3 min, followed by a rinse using distilled water, and returned to blue using a blue-returning solution. The coverslip was placed after an additional rinsing. ImageJ software was utilized to detect the AOD of positive areas.
Hematoxylin-eosin (HE) staining was performed using the correspondent kit (Solarbio, cat#G1120). Treated patient tumor and normal tissue sections were incubated in sufficient Hematoxylin solution for 5 min, rinsed twice using distilled water to remove the excess staining, and returned to blue using a blue-returning solution. The sections were rewashed with distilled water and dehydrated, then stained with eosin solution for 5 min. The sections were rinsed three times with absolute ethanol, placed in neutral gum, and the morphology was observed under an optical microscope.
Hematoxylin-eosin (HE) staining was performed using the correspondent kit (Solarbio, cat#G1120). Treated patient tumor and normal tissue sections were incubated in sufficient Hematoxylin solution for 5 min, rinsed twice using distilled water to remove the excess staining, and returned to blue using a blue-returning solution. The sections were rewashed with distilled water and dehydrated, then stained with eosin solution for 5 min. The sections were rinsed three times with absolute ethanol, placed in neutral gum, and the morphology was observed under an optical microscope.
3
Histological analysis of AV tissue
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All the AV tissues were dehydrated, embedded in paraffin, and cut in 5-µm-thick sections. The sections were deparaffinized with xylene and hydrated with gradient alcohol. For haematoxylin and eosin stain, sections were stained with hematoxylin (G1120, Solarbio) for 1 min and then with eosin for 1 min. For Alizarin red S staining, sections were stained with Alizarin Red S staining working solution (G3280, Solarbio) for 5 min and hematoxylin for 1 min. For immunohistochemical staining, after antigen retrieval with sodium citrate, sections were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase and blocked by 5% normal goat serum (SP-9000, ZSGB-BIO) for 15 min, sections were incubated with primary antibodies against OPN and NE overnight at 4 ℃.Then sections were further incubated with biotin-labeled goat anti-rabbit IgG for 15 min and HRP-labeled Streptomyces ovoalbumin working solution for 15 min. The signal was revealed by using a DAB Substrate Kit (ZLI-9017, ZSGB-BIO).
4
Quantitative Analysis of Tumor Angiogenesis
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After tumor removal, the fresh tumors were immediately embedded in optimum cutting temperature compound (Sakura, Zoeterwoude, Netherlands) and sectioned (5 mm), followed by staining with hematoxylin and eosin (H&E), CD31 antibody, and oil red O. Briefly, sections mounted on slides were dehydrated in ethanol, rinsed in PBS containing Tween 20 (PBST), and incubated with 0.3% hydrogen peroxide for 15 min. After washing with PBST, sections were blocked by incubation in 3% bovine serum albumin for 30 min, followed by overnight incubation with primary antibody (rabbit anti-CD31 diluted 1 : 50; Abcam). Slides were washed with PBST followed by a 1 h incubation with HRP-conjugated goat anti-rabbit secondary antibody (1 : 4000, Abcam), rinsed in PBST, and exposed to 3,3′-diaminobenzidine (Solarbio, Beijing, China). Then, counterstaining was performed with hematoxylin (Solarbio, Beijing, China). For H&E staining, sections were stained in hematoxylin for 3 min, washed in water, and then exposed for 5 min to eosin (Solarbio). The immunostaining results were analyzed using imaging software (Olympus, Tokyo, Japan).
5
RNA In Situ Hybridization for ZFAS1 Expression
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RNA ISH assay was performed strictly following the kit instructions (Boster, Wuhan, China). The sections were deparaffinized, deproteinized, and pre-hybridized with pre-hybridization solution at 42 °C for 2 h, then incubated with the DIG-labeled probe solution (Dilute 4 times with 1× PBS) at 37 ℃ overnight. The specific sequences of probes for ZFAS1 were showed in Additional file 1 : Table S14. After stringent washing, the slides were exposed to a streptavidin-peroxidase reaction system and stained with DAB (Zsbio, Beijing, China) for 2 min. Then 0.1% Hematoxylin (Solarbio, Beijing, China) was used to counterstain the slides for 5 min. ZFAS1 expression levels were observed and counted under a microscope (Nikon, Tokyo, Japan).
6
In situ Hybridization for ZFAS1 Expression
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In situ hybridization was performed strictly following the kit instructions (Boster, Wuhan, China). Before prehybridized in prehybridization solution at 42 °C for 2 h, slides were deparaffinized and deproteinated, then incubated with a digoxin-labeled probe solution (Dilute 4 times with 1 × PBS) at 37 °C over night (Specific probe sequences were shown in Table S5 ). After stringent washing, the slides were exposed to a streptavidin- peroxidase reaction system and stained with DAB (Zsbio, Beijing, China) for 2 min. Then 0.1% Hematoxylin (Solarbio, Beijing, China) was used to counterstain the slides for 5 min. ZFAS1 expression levels were observed and counted under a microscope (Nikon, Tokyo, Japan), and the evaluation analysis was described in Addition file 2.
7
In situ Hybridization for lncRNA ZFAS1
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In situ hybridization was performed strictly following the kit instructions (Boster, Wuhan, China). Before prehybridized in prehybridization solution at 42°C for 2h, slides were deparaffinized and deproteinated, then incubated with DIG-labeled probe solution (dilute 4 times with 1×PBS) at 37°C overnight (specific sequences of probes for ZFAS1 were showed in Supplementary Table 7 ). After stringent washing, the slides were exposed to a streptavidin-peroxidase reaction system and and stained with DAB (Zsbio, Beijing, China) for 2 min. Then 0.1% Hematoxylin (Solarbio, beijing, China) was used to counterstain the slides for 5 min. LncRNA expression levels were observed and counted under a microscope (Nikon, Tokyo, Japan).
8
Histopathological Evaluation of Lung Injury
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Paraffin-embedded sections were deparaffined, dehydrated, stained with hematoxylin (Solarbio) for 3 min, washed, and differentiated in 1% acid alcohol for 15 s. Next, sections were stained with eosin (Solarbio) for 2 min. Afterwards, the lung morphology was observed under an optical microscope (Olympus). As previously described, pathologists scored lung tissue damage according to a scale of 1 (no damage) to 4 (most severe).
After de-paraffining and dehydration, apoptosis was determined using the TUNEL assay kit (Nanjing Keygen Biotech CO., Ltd., Nanjing, Jiangsu, China). Next, cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology Co. LTD, Shanghai, China). The TUNEL positive cells were observed by means of fluorescence microscopy (Nikon, Tokyo, Japan). The apoptosis degree was evaluated by the ratio of TUNEL positive cells within the visual field and the total cells.
After de-paraffining and dehydration, apoptosis was determined using the TUNEL assay kit (Nanjing Keygen Biotech CO., Ltd., Nanjing, Jiangsu, China). Next, cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology Co. LTD, Shanghai, China). The TUNEL positive cells were observed by means of fluorescence microscopy (Nikon, Tokyo, Japan). The apoptosis degree was evaluated by the ratio of TUNEL positive cells within the visual field and the total cells.
9
Tissue Fixation and Histological Staining
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The tissue was fixed with 4% (w/v) paraformaldehyde (Sinopharm, Beijing, China) at room temperature overnight, rinsed with waster for 4 h, and dehydrated with graded concentrations of ethanol (70% (v/v) for 2 h, 80% for 2 h, 90% for 2 h and 100% for 1 h twice). Then the tissue was hyalinized with xylene (Sinopharm) for 30 min, paraffin-embedded, and cut into sections of 5 μm.
After drying on glass slide at 60 ℃, the sections were dewaxed in xylene (Sinopharm) for 15 min D r a f t twice, and rehydrated with 100%, 95%, 85% and 75% ethanol. Thereafter, the sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min, soaked in 1% (w/v) hydrochloric acid/ethanol for 3 s, and stained with eosin (Sinopharm) for 3 min. Afterwards, the sections were dehydrated with ethanol of 75%, 85%, 95% for 2 min each, and 100% for 5 min twice, and hyalinized with xylene for 10 min twice. After removing the residual liquid, the sections were mounted with half one drop of gum, dried at room temperature, and photographed with a microscope (Olympus, Tokyo, Japan) at 200× magnification.
After drying on glass slide at 60 ℃, the sections were dewaxed in xylene (Sinopharm) for 15 min D r a f t twice, and rehydrated with 100%, 95%, 85% and 75% ethanol. Thereafter, the sections were stained with hematoxylin (Solarbio, Beijing, China) for 5 min, soaked in 1% (w/v) hydrochloric acid/ethanol for 3 s, and stained with eosin (Sinopharm) for 3 min. Afterwards, the sections were dehydrated with ethanol of 75%, 85%, 95% for 2 min each, and 100% for 5 min twice, and hyalinized with xylene for 10 min twice. After removing the residual liquid, the sections were mounted with half one drop of gum, dried at room temperature, and photographed with a microscope (Olympus, Tokyo, Japan) at 200× magnification.
10
Histological Analysis of Perirenal Fat
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Perirenal fat were fixed with 4% paraformaldehyde and embedded in paraffin. For HE staining, sections were stained with hematoxylin (Solarbio, Beijing, China). Then, sections were photographed by the BX-50F light microscope (Olympus, Tokyo, Japan).
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