SOP: Cartilage Staining Procedures

REVISED: 6-19-2011

BACKGROUND/OBJECTIVE: Cell staining is a technique that can be used to visualize cells and cell components under a microscope. The main constituents of articular cartilage include aggrecan and type II collagen, both of which are proteolyzed when cartilage degenerates. Aggrecan, the major proteoglycan in cartilage, is a macromolecule consisting of an approximately 210-kDa core protein bound to a large number of sulfated chondroitin-side chains and multiple sulfated keratin-side chains. The core protein also has a domain capable of binding to hyaluronate produced by synoviocytes. Type II collagen is a fibrillary element responsible for maintaing the special mesh structure in healthy cartilage and forms the structure that takes into its pore water-retentive proteoglycan. Type II colagen, unlike type I and III, exhibits a foreighn-body antigenicity. Anti-type II collagen antibody, once generated in the body, induces rheumatisim or arthritis. To detect aggrecan and type II collagen in cartilage, Toludine blue, Safranin-O and Fastgreen stains can be used. Toludine blue and safranin-O are cationic stains (basic dyes) that stain acidic proteoglycan present in cartilage tissues. Toludine blue stains the cytoplasm light blue, and nuclear regions dark blue, and mast cells purple. Safranin-O binds to glucosaminoglycan (GAG) an orange colora and is often used to stain articular cartilage. Fast green, the contrast stain of Safranin-O, is a sulfate group containing acidic substrate, which binds strongly to the amino group on protein and thereby strongly stains non-collagen sites. A collagen type II antibody can be used to detect type II collagen by immunostaining. RESPONSIBILITY:It is the responsibility of the Principle Investigator and graduate students to ensure all laboratory personnel are properly trained in and follow this SOP. SAFETY: All laboratory personnel should comply with applicable MSDS and current health and safety regulations when working in the laboratory. Protective equipment such as disposable gloves must be worn when working in the lab. ABBREVIATIONS AND DEFINITIONS NBF 10% Neutral Buffered Formalin PBS Phosphate Buffered Saline GAG Glycoaminoglycan REFERENCES: Histology Staining Manual. Toludine Blue. <http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/TOLUID.PDF> Safranin O staining protocol for Cartilage. <http://www.ihcworld.com/_protocols/special_stains/safranin_o.htm> Cartilage staining kit. <http://catalog.takara-bio.co.jp/en/PDFFiles/MK310_e.pdf> 10% Neutral Buffered Formalin <http://www.bbcus.com/uploads/files/10pctNeutralBufferedFormailin_procedure.pdf>

SOP: Cartilage Staining Procedures 1. MATERIALS REQUIRED 1.1. EQUIPMENT a) Slides b) Microscopes 1.2. REAGENTS a) NBF b) 1% Acetic acid c) Toludine Blue 0.1% Toludine Blue Solution (pH 4.1) d) Safranin O 0.1% Safranin O solution e) Collagen type II antibody f) Diluent for antibody

REVISED: 6-19-2011

[Sigma-Aldrich F5304] [Sigma-Aldrich T3260] [Sigma-Aldrich S8884]

2. PREPARATION 2.1. DOCUMENTATION a) Document date reagents are prepared and expiration 2.2. PREPARING SLIDE a) Premeablization treatment of cells, generally with a mild surfactant, which dissolves cell membranes in order to allow larger dye molecules to enter inside the cell. b) Fixation serves to fix or perserve cell or tissue morphology through the preparation process. c) Mounting involves attaching samples to a glass microscope slide for observation and analysis. Cells may either be grown directly to the slide or loose cells can be applied to a slide using sterile technique. d) Staining This process involves immersing the sample (before or after fixation or mounting) in a dye solution and then rinsing and observing the sample under a microscope. 2.3. PREPERATION OF TOLUDINE BLUE-Oa) Stock Solution: 1g Toludine Blue -O- in 100ml 70% alcohol. Stable for 6 months. b) Working solution: 5ml Toludine blue stock solution to 45ml PBS or 1% sodium chloride. Make fresh, discard after use. 2.4. PREPERATION OF SAFRANIN-Oa) Safranin stock solution dissolve 2.5g safranin dye in 100ml of 95% ethanol. Note: It is easier to dilute a 1% solution to 0.1% solution so in general, the working solution should be 1g of dye to 100ml solvent. b) 0.1% Safranin O Solution Dissolve 0.1g of Safranin in O into 100 ml of distilled water 3. PROCEDURE 3.1. CELLS IN CULTURE a) Culture cells on a sterile slide in a sterile glass-petri dish. b) Gently remove the culture media c) Cover the slide with fixative solution and let stain at room temperature for 10 minutes. 2

SOP: Cartilage Staining Procedures

REVISED: 6-19-2011

d) Dilute the fixation with PBS, and remove the diluted fixative. Repeat this twice to wash the slide. e) Add 1% Acetic acid to each well, allow to react for 10-15 seconds and then remove the 1% Acetic acid. f) Add stain solution (Toludine Blue, Safranin O, Fast Green) and let stand at room temperature for 5 minutes to stain the cells. g) Wash 3 times with sterile PBS. Adjust the color level by altering the number of washes. h) Microscopic observation may be done in the presence of PBS. 3.2. TISSUE SECTION STAINING a) Prepare frozen sections or paraffin sections. Paraffin sections require deparafinization.

b) Add 100-200l of fixation solution to each tissue section and let stand at room temperature for at least 3 hours prior to processing. c) It is not necessary to wash the tissue after fixation. d) Add 1% Acetic acid to each tissue section, allow to react for 10-15 seconds and thn remove acetic acid. e) Add stain solution (Toludine Blue, Safranin, or fast green) to each tissue section and let stand at room temperature for 5 minutes to stain the tissue. Remove stain solution. f) Add absolute ethanol for destaining. Destain until the color density reaches appropriate level. g) Perform dehydration, clearing, and mounting. h) Note: Adjust the volume of fixation solution and stain solution depending on the size of the tissue. 4. EXPECTED RESULTS 4.1. FAST GREEN Contrast stain of Safranin-O, binds strongly to amino group on protein, strongly staining non-collagen sites 4.2. SAFRANIN O Binds to GAG, stains orange 4.3. TOLUDINE BLUE Stains acidic proteoglycan present. 5. NOTES 5.1. REAGENT STORAGE 3

SOP: Cartilage Staining Procedures

REVISED: 6-19-2011

a) Stain solutions are stable for up to 1 month at 4C. For long-term storage, dispense them into required aliquots and store at -20C. 5.2. EXPIRATION DATES a) TOLUDINE BLUE -O- STOCK SOLUTION: 6 MONTHS 5.3. SAFETY a) TOLUDINE BLUE Wear gloves, goggles, and lab coat. Avoid contact and inhalation. b) SAFRANIN O Wear gloves/eye protection/face protection 5.4. MSDS a) SIGMA T3260 TOLUDINE BLUE -Ob) SIGMA S8884 SAFRANIN O c) SIGMA F5304 10% NEUTRAL BUFFERED FORMALIN WITH 0.03% EOSIN