In total, 129 synovial samples were preferentially obtained from grossly inflamed (dull and opaque) synovium. If no inflammation was apparent, samples were obtained from standard locations: the femoral aspects of the medial and lateral gutters, and the central supratrochlear region in the suprapatellar pouch. Tissue samples were snap-frozen and stained with Harris’ modified hematoxylin solution and eosin Y (both from Sigma-Aldrich). Twenty histologic features (synovial lining hyperplasia, lymphocytes, plasma cells, Russell bodies, binucleated plasma cells, fibrin, synovial lining giant cells, sublining giant cells, fibrosis, detritus, necrosis, granulation tissue, neutrophils, mast cells, eosinophils, synovial chondrometaplasia, germinal centers, mucin, infarction, and vascularity) were scored using a systematic approach (as outlined in detail in Supplementary Methods , available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40428/abstract and also available at www.hss.edu/pathology-synovitis ). In total, 129 synovial samples were scored by a single pathologist (EFD), and 40 samples were scored again by a second pathologist (EG) to determine interrater reliability.
Harris modified hematoxylin solution
Manufactured by Merck Group
Sourced in United States
Harris modified hematoxylin solution is a laboratory reagent used in histology and cytology for the staining of cell nuclei. It is a blue-black nuclear stain that is commonly used in conjunction with eosin as a counterstain. The solution contains hematoxylin, a natural dye derived from the heartwood of the Logwood tree, which binds to the basic components of cell nuclei, producing a distinctive color.
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Lab products found in correlation
Permount, by Thermo Fisher Scientific (3 mentions)
Eosin y, by Merck Group (2 mentions)
Peroxidase donkey anti chicken antibody, by Stratech Scientific (2 mentions)
Dab substrate kit, by Vector Laboratories (2 mentions)
Ab13970, by Abcam (2 mentions)
Eosin y solution, by Merck Group (1 mentions)
Diaminobenzidine solution, by Merck Group (1 mentions)
Anti gfap antibody, by Thermo Fisher Scientific (1 mentions)
Biotinylated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Tris hcl buffer, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Xylene, by Merck Group (1 mentions)
Eosin solution, by Merck Group (1 mentions)
Optiphot transmitted light microscope, by Nikon (1 mentions)
Bouin s solution, by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Diaminobenzidine dab substrate kit, by Vector Laboratories (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Rm2145 microtome, by Leica (1 mentions)
13 protocols using harris modified hematoxylin solution
1
Comprehensive Synovial Tissue Histopathology
2
Histological Examination of Decalcified Bone
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The bone samples were decalcified using 5 % nitric acid for 48 h. The right and left parietal bones were separated through the midline sagittal suture. Both segments were embedded to show the sagittal sections in the paraffin blocks. Then, the sections were sliced and stained with hematoxylin and eosin (H&E).
Paraffin-embedded tissue blocks were sliced to a thickness of 5 μm. The sections of each tissue were carefully placed on silane-coated slides. These slides were incubated at 60 °C for 24 h. After cooling at room temperature, the tissue slides were soaked in 100 % xylene for 5 min in triplicate. The tissue sections were then hydrated by the consecutive application of high- to low-grade ethyl alcohol. Fully hydrated tissue sections were washed with distilled water. After that, tissue sections were stained with Harris modified hematoxylin solution (Sigma Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Then, de-staining was performed with 1 % acid alcohol for 1 s. De-stained tissue sections were washed in running tap water for 10 min. Next, Eosin Y solution (Sigma Aldrich, St. Louis, MO, USA) was applied on the tissue sections for 1 min. Then, after gradational hydration with ethyl alcohol and clearing with xylene, the tissue sections were fixed by paramount solutions.
Paraffin-embedded tissue blocks were sliced to a thickness of 5 μm. The sections of each tissue were carefully placed on silane-coated slides. These slides were incubated at 60 °C for 24 h. After cooling at room temperature, the tissue slides were soaked in 100 % xylene for 5 min in triplicate. The tissue sections were then hydrated by the consecutive application of high- to low-grade ethyl alcohol. Fully hydrated tissue sections were washed with distilled water. After that, tissue sections were stained with Harris modified hematoxylin solution (Sigma Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Then, de-staining was performed with 1 % acid alcohol for 1 s. De-stained tissue sections were washed in running tap water for 10 min. Next, Eosin Y solution (Sigma Aldrich, St. Louis, MO, USA) was applied on the tissue sections for 1 min. Then, after gradational hydration with ethyl alcohol and clearing with xylene, the tissue sections were fixed by paramount solutions.
3
Synovial Tissue Histological Scoring
4
H&E Staining of JB-4 Plastic Sections
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H&E staining of JB-4 plastic sections was carried out as described previously [38 (link)] with the following modifications: Acid and base washes were omitted, Harris Modified Hematoxylin Solution (Sigma) and Eosin Y (EMD) were used, and tap water replaced prescribed substitute. Slides were air dried for 15 mins [39 (link)] and coverslipped using Permount (Fischer Scientific). Myocardial ventricle wall thickness for each embryo was the average of measurements made at two points along the anterior–posterior axis from 1–2 sections stained by H&E.
5
Immunohistochemical Analysis of GFP
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Air dried cytospins were fixed with 4% PFA in PBS for 5 minutes. Slides were first blocked with PBS/5%BSA containing Fc-block for 30 min at RT and then with 3% H2O2 in water for 5 minutes. Slides were incubated with chicken polyclonal anti-GFP (Abcam, ab13970) in PBS/5% BSA for 4 hours at room temperature and next with peroxidase donkey anti-chicken antibody (Stratech, 703-035-155-JIR) for 1 hour at room temperature. Peroxidase was detected with DAB substrate kit (Vector Laboratories, SK-4100) according to manufacturer’s instructions. Cells were counterstained with Harris Modified Hematoxylin Solution (Sigma-Aldrich, HHS32)) for 10 minutes.
6
Hematoxylin and Eosin Staining of Plastic Sections
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Hematoxylin and eosin staining of plastic sections was carried out in accordance with the protocol described previously,24 (link) with the following modifications. Acid and base washes were omitted, Harris modified hematoxylin solution (Sigma-Aldrich Corp., St. Louis, MO, USA) and eosin Y were used, and regular tap water was used instead of the prescribed substitute. Slides were air-dried for 15 minutes and coverslipped using Permount (Fischer Scientific).
7
Astrocyte Marker Expression Analysis
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Immunohistochemical staining was used to analyze the expression of glial fibrillary acidic protein (GFAP), an astrocyte marker, in the sections of the anterior horn. First, the tissue section slides were deparaffinized in xylene (Merck, Darmstadt, Germany) and the slides were immersed in ethanol (Merck) for rehydration. Afterwards, the tissue slides were transferred to a 10 mM sodium citrate buffer (Sigma) at pH 6.0 for antigen retrieval. The endogenous peroxidase was blocked by tissue incubation in 3% hydrogen peroxide. Among incubations, two washes were performed with Tris/HCl buffer (Sigma) at pH 6.0. Next, tissue samples were incubated with rabbit polyclonal anti-GFAP antibody (1:250; Invitrogen, Carlsbad, CA, USA) with immunoglobulin overnight at 4°C and then with biotinylated goat anti-mouse antibody (1:200; Invitrogen) for 2 hours at room temperature according to manufacturer instructions. Visualization of immunoreactivity was achieved after incubation of the tissue sections in diaminobenzidine solution (0.1%; Sigma). Finally, counterstaining was performed using Harris-modified hematoxylin solution (Sigma).
8
Hematoxylin-Eosin Staining of Prostate Tissue
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Paraffin-embedded prostate tissue sections were deparaffinized and rehydrated following conventional methods. Harris modified hematoxylin solution (Sigma) was applied to sections for 5 min followed by water washing. Then 1% Eosin solution (Sigma) was applied to sections for 3 min. After thorough wash, sections were mounted using neutral balsam.
9
Hematoxylin-Eosin Staining Protocol
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Cells were fixed with Bouin’s solution (Sigma–Aldrich, St Louis, MO, USA) (RT, 30 min), followed by washing with 70% ethanol (20 min) and double-distilled water (ddH2O). Cells were stained with Harris modified hematoxylin solution (Sigma–Aldrich, St Louis, MO, USA) (RT, 20 min), followed by washing with ddH2O and 70% ethanol. Cells were stained with 1% eosin (British Drug Houses, Ltd., London, UK) in 70% ethanol (RT, 7 min). Cells were washed twice (5 min each) with ethanol (70%, 96%, 100%) and xylol. Entellan® mounting fluid (Merck, Darmstadt, Germany) was used to mount coverslips onto slides. A Nikon Optiphot transmitted light microscope (Tokyo, Japan) was used for visualization. The cells were scored blind, with slides deidentified, and were only linked to each treatment condition after the analysis was concluded.
10
Immunohistochemical Analysis of GFP
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