Materials & Methods For Chemicals Reagents

Tris Buffered Saline (TBS)

Tris solution for zinc fixation : 2.1 g of Trizma base was dissolved in 1000ml of distilled water with the help of magnetic stirrer and stirrer bar. This solution was then sterilised by autoclaving and then stored for a month at room temperature.

Tris Buffered Saline for Immunohistochemistry :242.26 g of Trizma base was dissolved in 500ml of distilled water with the use of magnetic stirrer and stirrer bar. The pH of 7.6 was adjusted and autoclaved for sterilization and then was stored at room temperature for a month.

Phosphate Buffered Saline (PBS)

Five tablets of PBS were dissolved in 500ml of distilled water. The pH was then adjusted between 7.4-7.6. This solution was then autoclaved and stored at room temperature.

Antibody diluent

6ml of sodium azide and 300µl of bovine serum albumin was added in 100ml of TBS and mixed with magnetic stirrer. The pH of 7.6 was adjusted and stored for three months at 40C.

Cleaning of glassware

1% (v/v) detergent (Neutracon®, Decon Laboratories Ltd) solution free from phosphate was used. Glassware like beakers and bottles (100ml, 1,000ml and 2,000ml) were immersed overnight for cleaning. The glass wares were first rinsed with tap water and then with distilled water to remove all the residues of detergent. These were then dried and sterilised at 180oC for 4hrs as required using dry heat.

pH measurement

Standard pH solutions found at pH 4, 7 and 10 were used to calibrate the pH meter. Magnetic stirrer plate was used to place the solutions and stirred gently with stirrer.

Light microscopy

Olympus BX51 microscope was used to carry out bright-field microscopy. It was adjusted at position 1 using the BX-RFA dial. Images were captured with Olympus XC50 digital camera. Cell B software (Olympus, UK) was used to display digital images.

Fixation

Before decalcification, all tissue samples were fixed in 30 ml 10% (v/v) neutral buffered formalin (NBF) and zinc for 48 hrs.

Histological techniques

• Paraffin wax embedding: Plastic cassettes were used and bovine cartilage of 6mm in size was placed on them. These were cycled in the Lecia TP120 automated tissue processor. Further processing of these tissues was done by immersing them in ethanol of 70% (v/v) for 1hr.
• Tissue sectioning: The hotplate was set at 550C and water bath at 450C. Both were switched on and left for 10 minutes to reach particular temperatures. The section thickness was selected at 5 µm and angle of blade was at 50 angles. Sections were removed using fine tip forceps and hair brush and placed in water bath by which wrinkles within the sections were removed. Sections were then captured by placing the Superfrost Plus Slides underneath the floating sections followed by lifting towards surface of water.
• De waxing and section hydration: Slides were placed in xylene for 10 minutes and again placed in fresh xylene for 10 minutes for the purpose of dewaxing, The sections were dehydrated by immersing them in 100% (v/v) ethanol for 3 minutes, 2 minutes and 1 minute successively.
• Dehydration of sections: The sections were placed in 70% (v/v) ethanol for 5 seconds and immediately immersing in 100% (v/v) ethanol for 1, 2 and 3 minutes concluding the dehydration process. The slides were kept in xylene (2x) for 10 minutes.
• Haematoxylin & Eosin (H&E) staining: H&E staining was used to determine the native construction of the bovine cartilage tissue prior and subsequent to the experimentation. Dewaxing and rehydration of slides was carried out and was immersed in haematoxylin for a minute.

Immunohistochemistry

Tissue fixation:

The tissue underwent zinc and NBF fixation for 24 hours. They were kept overnight (11 hours) in the tissue processor on program 1. The tissue was embedded and sectioned as previously mentioned following which the tissue samples were kept for 20 minutes on hot plate. For drying, these samples were placed overnight on a drying rack.

Antigen retrieval method:

The tissue samples were circled in order to use a smaller quantity of antigen retrieval solution with the help of lmmEdge hydrophobic barrier pen. Tissue samples were subjected to proteinase K antigen retrieval solution (DAKO, 53020) at room temperature for 20 minutes. They were then placed on a non-moving orbital shaker.

Immunohistochemistry method:

Dewaxed and rehydrated tissue sections were outlined with a hydrophobic pen and these tissues were subjected to proteinase K for 20 minutes. The slides were washed for 20 minutes using 3% (v/v) hydrogen peroxide (30% (v/v) hydrogen peroxide diluted to a 1:10 ratio with dPBS) solution followed by 3 minutes immersion in water.

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