To evaluate myofibrillar loss, GM sections were stained with haematoxylin and eosin as we described previously.21 , 22 Sections were deparaffinized, rehydrated, and subsequently stained with haematoxylin (Thermo Fisher Scientific; cat#7211), 1% acid alcohol (Poly Scientific R&D Corp; cat#S104), bluing reagent (Thermo Fisher Scientific; cat#73011), and eosin (Thermo Fisher Scientific; cat#7111) solutions. Nuclei were stained in blue/purple, and muscle cells were in pink. Images were recorded using Keyence microscope (Itasca, IL, USA). ImageJ software was used to quantify myofibrillar size (mm2) at ×20 magnification captured pictures, and data were represented in a bar graph using SigmaPlot software. Representative images were recorded at ×40 magnification.
Haematoxylin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Haematoxylin is a natural dye derived from the Logwood tree. It is commonly used as a stain in histology and cytology laboratories to visualize cellular structures and components under a microscope. Haematoxylin stains cell nuclei blue, providing contrast to highlight cellular details for analysis and examination.
Automatically generated - may contain errors
Lab products found in correlation
Eosin y, by Thermo Fisher Scientific (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Species isotype, by Santa Cruz Biotechnology (2 mentions)
A21434a21436 s32355, by Thermo Fisher Scientific (2 mentions)
Vectasheld mounting media, by Vector Laboratories (2 mentions)
A21448, by Thermo Fisher Scientific (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Bluing reagent, by Thermo Fisher Scientific (1 mentions)
Fast green solution, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Abcam (1 mentions)
Aperio at2 system, by Leica (1 mentions)
Abc peroxidase standard staining kit, by Thermo Fisher Scientific (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Poly horseradish peroxidase solution, by Immunologic (1 mentions)
Superblock solution, by Thermo Fisher Scientific (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Differentiation rtu, by Avantor (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
Slide scanner, by 3DHISTECH (1 mentions)
22 protocols using haematoxylin
1
Quantifying Myofibrillar Size in GM Sections
2
Histological Assessment of Intervertebral Disc Degeneration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure the degree of IVDD, all rats were sacrificed at 8 weeks after surgery. After tissue collection, tissue sections (5 µm) were carefully cut. In addition, the disc tissue sample sides were carefully stained by Safranin O fast green (SO), Haematoxylin and eosin (H.E) and Alcian Blue (A.B), according to the manufacturer’s protocols. For SO staining, deparaffinized sections were stained with SO solution (Sigma-Aldrich) and subsequently counterstained with 0.2% fast-green solution (Sigma-Aldrich). For HE staining, deparaffinized sections were stained with Haematoxylin (Thermo Scientific, UK) for 7 min and counterstained in Eosin (Thermo Scientific, UK) for 1 min. A.B staining was performed using Alcian Blue solution (0.1% A. B 8GX in 0.1 N HCl) for 30 min at room temperature. H. E staining was performed to evaluate the morphological changes of NPCs. The SO staining and A. B staining were performed to detect the cellularity and morphology of NP tissues examined by other three histology analysts in a blinded manner by a light microscope and carefully evaluated by using a grading scale, as described previously [34] (link). Therefore, the histolopathological scores were 5 for the normal disc, 6–11 for the moderate degenerated disc and 12–15 for the severe degenerated disc. In the end, images were scrupulously captured using a light microscope (Nikon, Japan).
3
Immunohistochemical Analysis of Paraffin-Embedded Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded tissue samples were cut into 5 µm sections, deparaffinized and rehydrated. For antigen retrieval, slides were boiled in citrate buffer (0.01 M, pH 6.0) using a microwave oven on high power for 5 min and cooled down to room temperature. After incubation in 3% aqueous H2O2 to quench the endogenous peroxidase, the sections were washed in PBST (PBS with 0.1% Tween-20, v/v) washing buffer, blocked with 5% goat serum (EMD Millipore) diluted in PBST at room temperature for 1 h and incubated with primary antibodies diluted in 2% goat serum in PBST at 4 °C overnight.
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table3 ) for 1 h, and then processed according to the ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min. The slides were stained with 3,3′-diaminobenzidine (Abcam) for 5 s to 5 min and counterstained with haematoxylin (Thermo Fisher Scientific) for 45 s. The images were scanned using the Leica Aperio AT2 system at Stanford Human Pathology/Histology Service Center. Serial sections were incubated with GS and MYC-tag primary antibodies, and a cluster of cells (at least 20 cells) positive both for GS and MYC-tag were counted as early foci. The antibodies used in this study are shown in Supplementary Table 3 .
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table
4
Cardiac Tissue Cardiomyocyte Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The blind sample analysis of the cardiomyocyte sizes from the cardiac apex was carried out as described recently66 . In brief, after rinsing the cardiac tissue with phosphate-buffered saline (PBS) and dehydration, samples were embedded in PEG 1500 (Merck-Schuchhardt, Hohenbrunn, Germany). Thereafter, sections of 2 µm thickness were prepared (Microm HM 335 E, Thermo Fisher Scientific, Waltham, MA, USA) and stained with haematoxylin (Thermo Fisher Scientific) prior to analysis of myofibril area (Axiovert 200 microscope, AxioCamMRc, Axiovision Rel. 4.8.2 software).
5
Optimizing Antigen Retrieval for Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody specificity and optimal conditions for antigen retrieval were assessed by singleplex IHC. FFPE 4‐μm tissue sections were deparaffinised with xylene and washed in ethanol, after which endogenous peroxidase was blocked by incubation in a 0.3% hydrogen peroxide/methanol (Merck Millipore, Burlington, MA, USA) solution for 20 min. Heat‐induced antigen retrieval was done with either citrate buffer (10 mm , pH 6.0) or Tris–EDTA buffer (10 mm /1 mm , pH 9.0). After cooling, non‐specific antibody binding sites were blocked for 30 min with Superblock solution (Thermo Fisher Scientific, Waltham, MA, USA) and incubated overnight with a primary antibody (Table 1 ). After washing in PBS, 1 h incubation with poly‐horseradish peroxidase solution (Immunologic, Duiven, The Netherlands) was performed at room temperature (RT). The slides were developed with the DAB+ chromogen (DAKO, Agilent Technologies, Santa Clara, CA, USA) solution and counterstained with haematoxylin (Thermo Fisher Scientific). Optimal IHC conditions were evaluated by light microscopy.
6
Histological Staining of Prostate Tissue
7
Histological Analysis of Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen sections (5 μm, cut using paraffin rotary microtome (Leica)) were stained with Haematoxylin (ThermoFisher) and Eosin Y (ThermoFisher) (H&E). Coverslips were applied using DPX mountant (Sigma, 06522). Spleen sections were stained for iron with Perls Prussian Blue Stain Kit (Abcam, 65692) according to manufacturer’s instructions. Sections were scanned on SlideScanner (3D Histech Panoramic P250) and analysed on CaseViewer (3Dhistech Ltd.).
8
Quantifying Angiogenesis in Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The slides with tumour tissues were immersed in peroxidase I blocking reagent (Biocare, Medical, USA) for 5 minutes and immersed in preheated Diva Decloaker (Biocare Medical, USA). They were heated in a microwave for 20 minutes. The tissues were then treated with rat anti-CD31 IgG2a (Dianova, Germany) at 4 °C overnight. After washing with PBS for 1 minute for 3 times, a rat-on-mouse-HRP-polymer kit (Biocare Medical, USA) containing rat-on-mouse-HRP-polymer and rat probe was applied according to the procedures recommended by the manufacturer. After washing the slides with PBS for 1 minute for 3 times, pre-warmed 3,3′-Diaminobenzidine (DAB) (Open Biosystems, USA) were added onto the tissues for 5 minutes at 55 °C, and the slides were then immersed with haematoxylin (Thermo Scientific, USA) for 30 seconds. After washing with tap water, they were immersed with 70% and 80% ethanol quickly with shaking. They were then dehydrated gradually in 90%, 100% ethanol twice for 2 minutes, followed by immersed with 100% xylene for 2 minutes for 3 times. The expression of CD31 was visualized on the sections, appeared as brown in colour. Several sections at 2 levels (500 μm between each level) of each tumour tissue were photographed (x100), and the number of CD31 expressed cells was counted per sections in a blinded-manner.
9
Immunohistochemical Analysis of Siglec-6 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry analysis was performed as previously described [7] (link). Briefly, 8 m formaldehyde fixed paraffin embedded (FFPE) tissue sections from NP were rehydrated and blocked in 10 % fetal calf serum (FCS) in PBS supplemented with 1 % 0.01M Tween-20 (PBS-T) for 1 hour. Tissue sections were incubated with polyclonal sheep anti-Siglec-6 (2 µg/ml in 1 % FCS in PBS-T, AF2859, R&D systems) at 4 °C overnight. Sections incubated with 1 % FCS with no antibody served as the negative control. Sections were then incubated with HRP-conjugated secondary antibody anti-sheep (1/200 in 10 % FCS in PBS-T, P0163, Dako Cytomation) for 1 hour at room temperature prior to visualisation with DAB (Vector laboratories) and counterstaining with haematoxylin (ThermoFisher).
10
Foam Cell Visualization via Oil Red O
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oil red O (ORO, Sigma Aldrich, USA) was used to assess and confirm the presence of foam cells in the spheroids. Lipid vacuoles present in foam cells would be stained by oil red O staining, and Haematoxylin was used to counterstain the nucleus. 0.4% w/v of ORO was diluted in isopropyl alcohol/IPA (2-propanol, Merck, Germany) and kept as stock solution. Before each staining, DI water was added into the stock solution to create a working solution at volume ratio of 60:40 stock solution and DI water respectively, and subsequently filtered through filter paper (Qualitative Filter Paper No. 1, Advantec, Japan) then 0.2 µm syringe filter (Minisart®, Sartorius AG, Germany). The samples were pre-wet in 60% IPA for 10 minutes and immersed in ORO working solution for 30 minutes at room temperature. Stained samples were rinsed with DI water twice and washed with 60% IPA twice for at least 5 minutes. Nuclear counterstain using Haematoxylin (Thermo Fisher Scientific, USA) were also done. Eventually, the samples were embedded in aqueous mounting medium (glycerol gelatin, Sigma Aldrich, USA), covered with glass cover slips, and let to rest overnight before imaged under a light microscope (IX53, Olympus, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!