The sections were thawed at room temperature for 30 min before the procedures of HE staining were performed. The procedures comprised mainly fixation of 4% paraformaldehyde, 3 min Harri’s hematoxylin staining (Harri’s hematoxylin, Cat# HHS80, Sigma), and 40 second eosin staining, and 20% Glycerin-aided (Glycerin, Cat# 3783.1, Carl Roth, Germany) coverslipping. Staining was applied only in the sections of the tissue blocks (the staining was avoided in the sections of three-targeted models since the targets had been labeled with various colors). Traditional HE staining protocol was adopted for the slides indicated in Table S7 , Supplementary Information.
Harris hematoxylin
Manufactured by Merck Group
Sourced in United States, Germany
Harris hematoxylin is a laboratory staining reagent used in histology and cytology. It is a nuclear stain that colors cell nuclei blue-purple, allowing for the visualization and identification of cells and their structures under a microscope. The core function of Harris hematoxylin is to provide a contrast stain for the observation and analysis of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Merck Group (8 mentions)
Vectastain abc kit, by Vector Laboratories (7 mentions)
Eosin y, by Merck Group (7 mentions)
Permount, by Thermo Fisher Scientific (6 mentions)
Nanozoomer 2.0 rs, by Hamamatsu Photonics (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (3 mentions)
Dpx mounting medium, by Merck Group (3 mentions)
Oct compound, by Sakura Finetek (3 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Caspase 3, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Rm2125rt, by Leica (2 mentions)
Eosin g, by Merck Group (2 mentions)
Goat anti rabbit igg h l hrp, by Abcam (2 mentions)
Diaminobenzidine dab, by Merck Group (2 mentions)
Canada balsam, by Merck Group (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
143 protocols using harris hematoxylin
1
Hematoxylin and Eosin Staining Protocol
2
Quantification of Apoptotic Pericytes in Retinal Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze ACs and PL, RTD preparations representing the three groups of rats were stained with PAS and Harris hematoxylin (Sigma-Aldrich, St. Louis, MO). The RTD slides were immersed in 0.5% PAS (Sigma-Aldrich) for 10 min, rinsed in dH2O, and exposed to Schiff's reagent (Electron Microscopy Sciences, Hatfield, PA) as previously described [10 (link)]. Next, the slides were rinsed in dH2O and immersed in Harris hematoxylin (Sigma-Aldrich) for 20 s. After rinsing in dH2O, the slides were subjected to dehydration through an ethanol gradient and clearing in xylene and then were mounted in mounting medium (Permount; Fisher Scientific, Pittsburgh, PA). Ten representative 36 μm × 27 μm images were photographed using a digital camera (DS-Fi1; Nikon, Tokyo, Japan) connected to a computer, and the images were analyzed for ACs and PL. Apoptotic pericytes were identified as PL (pericyte ghosts) based on prominent histological characteristics, including basement membrane protrusion as an empty shell. Capillaries devoid of pericytes and endothelial cells were considered ACs. Counts were also scored by a second independent examiner in a masked manner. The data represent the average of both scores.
3
Histological Analysis of Lung Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung sections (sham 0 and 12 and CLP 0 and 12, n = 3 in each group) were deparaffinized and hydrated. To examine their histological features, the lungs were stained with hematoxylin and eosin (H&E). After blocking of endogenous peroxidase, antigen retrieval was performed in a high-temperature Tris-citrate buffer (pH 7.2). The rabbit polyclonal anti-SOD3 (Santa Cruz Biotechnology) (diluted 1:1,600) was used as the primary antibody. The Vectastin ABC Kit (Vector Laboratories, Burlingame, CA, USA) was used as the secondary antibody, and 3,3-diaminobenzidine (DAB, Sigma, St. Louis, MO, USA) was used as the chromogen. Thereafter, the sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). In the negative controls, the first antibody was omitted from the procedure, and the tissues were incubated with bovine serum albumin (BSA) instead.
4
Apoptosis Detection in Pneumocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caspase-3 primary antibody (1:200, rabbit polyclonal, Caspase-3, Abcam, United Kingdom) and secondary antibody (Goat Anti-Rabbit IgG H&L-HRP, ab205718, Abcam, United Kingdom) kits were used to identify apoptotic pneumocytes.
Lung tissue sections were first subjected to deparaffinization, followed by antigen retrieval procedures as recommended by the manufacturer. They were then incubated for 60 min with primary and secondary antibodies. Lung tissue sections were finally stained with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St. Louis, Missouri, United States of America) and Harris hematoxylin (Merck, Germany).
Lung tissue sections were first subjected to deparaffinization, followed by antigen retrieval procedures as recommended by the manufacturer. They were then incubated for 60 min with primary and secondary antibodies. Lung tissue sections were finally stained with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St. Louis, Missouri, United States of America) and Harris hematoxylin (Merck, Germany).
5
Immunohistochemical Staining of CRC Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining of the CRC tissues was performed using a tissue microarray (TMA) block of 343 patient tissues. The tissue core punched by a 2-mm puncher (Unitech Korea Co., Ltd.) was embedded in a recipient paraffin block (Unitech Korea Co., Ltd.). The TMA blocks were cut into 4-µm sections, dewaxed in xylene and rehydrated through a gradient concentration of ethanol (100, 95, 90, 80 and 70%) for 5 min per concentration. Subsequently, the blocks were incubated in 0.01 M citrate buffer (pH 6.0) at 95°C for 30 min in a microwave. Endogenous peroxidase activity was inactivated using 0.3% H2O2 for 1 h at room temperature. The sections were subsequently incubated with antibodies against TET2 (1:100; cat. no. ab245287; Abcam) and AMPK (1:100; cat. no. GTX52341; GeneTex, Inc.) overnight at 4°C, followed by incubation with an anti-rabbit EnVision secondary antibody (cat. no. K4002; Dako; Agilent Technologies, Inc.) for 1 h at 37°C. For visualization, the sections were treated with µl 3,3′-diaminobenzidine solution (Dako; Agilent Technologies, Inc.) and counterstained with Harris' hematoxylin (EMD Millipore). The sections were mounted using Canada Balsam (Sigma-Aldrich; Merck KGaA).
6
Apoptosis Detection in Renal Tubules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caspase-3 (1:200, rabbit polyclonal Caspase-3, Abcam, UK) placed onto positively charged slides was used as the primary antibody for the determination of apoptotic renal tubule cells, together with kits containing an appropriate secondary antibody (goat anti-rabbit IgG H&L [HRP], ab205718, Abcam, UK).
Kidney tissue sections, 1-3 µm in thickness and cut using a microtome, were placed onto positively charged slides and subjected to deparaffinization and antigen retrieval procedures. Next, following incubation with primary and secondary antibodies, in line with the manufacturer’s instructions, 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO, USA) was applied. Finally, sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany).
Kidney tissue sections, 1-3 µm in thickness and cut using a microtome, were placed onto positively charged slides and subjected to deparaffinization and antigen retrieval procedures. Next, following incubation with primary and secondary antibodies, in line with the manufacturer’s instructions, 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO, USA) was applied. Finally, sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany).
7
Quantitative Immunocytochemical Analysis of β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The streptavidin–biotin method by using a Vectastain Elite Universal ABC kit (Vector Laboratories) was used for immunocytochemical staining [23 (link)]. Nonspecific binding sites in ethanol-fixed samples were quenched with 0.03% H2O2, normal horse serum, and using an Avidin D–Biotin blocking kit (Vector Laboratories). Subsequently, rabbit monoclonal XP β-catenin (1:100 dilution, Cell Signaling Technology; Clone ID: D10A8, cat. No. 8480S; Research Resource Identifier (RRID): AB_11127855) was used as the primary antibody. 3,3′-Diaminobenzidine (Vector Laboratories) was the chromogen and the samples were counterstained with Harris’ hematoxylin (Merck). Positive (HeLa cells; Figure 1A Figure 1B
A blinded evaluation (without the knowledge of the case) was conducted using a procedure described previously [23 (link)]. H-score was used to quantify the immunocytochemical staining results [24 (link)]. Staining intensity was also evaluated for each cell type, namely superficial, intermediate, and parabasal cells. Next, H-scores were calculated using a formula described in the literature [23 (link), 24 (link)].
A blinded evaluation (without the knowledge of the case) was conducted using a procedure described previously [23 (link)]. H-score was used to quantify the immunocytochemical staining results [24 (link)]. Staining intensity was also evaluated for each cell type, namely superficial, intermediate, and parabasal cells. Next, H-scores were calculated using a formula described in the literature [23 (link), 24 (link)].
8
Immunohistochemistry Protocol for Cell Marker Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed according to the method described by Camargo et al. [35 (link)]. The antibodies used for the markers and dilutions in this study are listed in Table 3 . Digestion of the antigen at high temperature was performed in a pressure cooker for 1 min using citrate buffer (6.0 pH). For incubation with the primary antibody, the sections were diluted in bovine serum albumin (BSA) solution and applied to each slide. Then, the slides were incubated overnight in a humid chamber at 4 °C for 18–22 h. The slides were then washed in phosphate-buffered saline (PBS) and incubated with the secondary antibody using the ABC Kit with Vectastain (Vector Laboratories, CA, USA) (Table 3 ). To visualize the positive cells, the slides were washed in PBS and proteins were visualized using diaminobenzidine (DAB) chromogen (70 mg DAB in 110 mL of the Tris-HCl; Sigma Chemical Co., St. Louis, MO, USA) and Harris hematoxylin (Merck, Darmstadt, Germany), and finally mounted with microscopy resin.
9
Histological Tissue Preparation and Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following an overnight incubation at 60 °C, slides were washed in xylene (twice × 5 min) then transferred to decreasing ethanol concentrations (100%, 96%, 70%), washed in double distilled water (DDW) (twice) and stained by Harris Hematoxylin (Merck KGaA, Darmstadt, Germany). After washing in tap water, slides were shortly dipped in 1% acid alcohol and consequently washed in water. Finally, a 1-min staining in 1% Eosin (Merck KGaA, Darmstadt, Germany) was performed, followed by washes in DDW, ethanol (70%, 96%, 100%) and cover slipping.
10
Histopathological Evaluation of NAFLD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The formalin-fixed liver tissues were embedded and sectioned at 3 µm thicknesses. The sections were then stained with Harris hematoxylin (Merck, Darmstadt, Germany) and eosin (Merck, Darmstadt, Germany) (H&E) for visualization of hepatic tissue architecture, morphological changes, and inflammatory cell infiltration. All histopathological examinations were graded according to the NALFD activity score (NAS) grading system for rodent [53 (link),54 (link)] by two independent pathologists, who were blinded to the experimental and serological data. A NAS score of ≥5 is diagnosed as NASH and a score of ≤3 is referred to as non-NASH. The stained sections were photographed using a light microscope (Olympus BX41, Tokyo, Japan) with a digital camera (Olympus XC50, Tokyo, Japan) at 40× magnification.
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!