STAINING METHODS

2.3.1 Histochemistry

The majority of routine histological processing was kindly performed by Ms C Noel and Mrs E Clayton, Histologists, RAFT Institute of Plastic Surgery. All specimen sections were de-waxed using xylene, rehydrated in decreasing concentrations of alcohol and then brought to water before the staining process began.

Haematoxylin and Eosin

Sections were stained with Mayer's haematoxylin for 10 minutes. They were then washed for a further 10 minutes in running tap-water. The stain was differentiated by placing the sections into acid-alcohol for 10 seconds (1% concentrated hydrochloric acid in 70% alcohol) and then "blued" in running tap-water for 5 minutes. After that the sections were placed into 1% eosin for 5 minutes and washed in running tap- water. Sections were dehydrated by running through increasing concentrations of alcohol, cleared in xylene and mounted with a coverslip. The results are: Nuclei -

Chapter 2 - M aterials and Methods

blue/black, Cytoplasm - varying shades o f pink, Muscle fibres - deep pinky red, Collagen - pale pink and Fibrin - deep pink

Modified SACPIC Technique (Nixon, 1993)

The sections were stained with celestine blue for 5 minutes and then rinsed in running tap-water. Next they were placed into Gill's haematoxylin for 5 minutes and then again rinsed in running tap-water. The stain was then "blued" in Scott's tap-water for 2 to 5 minutes and afterwards rinsed in running tap-water. The sections were placed into 2% saffanin for 5 minutes, rinsed in 70% ethanol which was followed by a further rinse in 95% ethanol. Differentiation occurred in absolute picric acid/ethanol for 3 minutes and the sections rinsed in 95% ethanol, then 70% ethanol and finally running tap-water. Further staining was performed in picro indigo carmine for 1 minute and then the sections rinsed in running tap-water. Dehydration through increasing alcohol concentrations then followed, the sections cleared in xylene and finally mounted with a coverslip. The results are: Nuclei - blue/black. Keratin - yellow. Collagen - blue. Inner root sheath - bright red. Outer border brush end - orange. Outer root sheath - pale green. Smooth muscle - green.

Massons Fontana

The sections were stained with a silver solution (10% silver nitrate with concentrated ammonia) and left overnight. They were then washed in distilled water and placed into 5% sodium thiosulphate for 2 minutes. After rinsing in running tap-water, the sections were stained in 1% aqueous neutral red for 3 minutes. After a final rinsing in running tap-water, the sections were dehydrated through increasing alcohol concentrations, cleared in xylene and mounted with a coverslip. The results are: Melanin - black. Argentaffin - black, Lipoftiscin - black. Nuclei - red.

Massons Trichrome

water. They were then stained with Mayer's haematoxylin for 5 minutes and afterwards washed until blue. Differentiation with 1% acid alcohol occurred for 10 seconds before washing in running tap-water. Staining in acid fuchsin solution for 5 minutes (acid fiichsin 0.5g, glacial acetic acid 0.5cm^ and distilled water 100 cm^) was then followed by rinsing in distilled water. The sections were then stained in phosphomolybdic acid solution for 5 minutes (phosphomolybdic acid 1 g and distilled water 100 cm^), drained and then stained with methyl blue solution for 2 to 5 minutes (methyl blue 2 g, glacial acetic acid 2.5 cm^ and distilled water 100 cm^). The sections were rinsed in distilled water and then treated with 1% acetic acid for 2 minutes before being dehydrated through increasing alcohol concentrations, cleared in xylene and mounted with a coverslip. The results are: Nuclei - blue, Muscle - red. Fibrin - red and Collagen - blue.

2.3.2 Immunohistochemistry

Detection of p53 and Heat Shock Protein 70 (HSP 70) Expression in Human Tissue

After de-waxing, the slides were placed into a plastic slide rack in a bath containing 250 ml of 10 mM citric acid at pH 6. The sections were microwaved three times for 4 minutes at 800 Watts. During this time the solution was topped-up with distilled water at the intervals. The sections were left to stand for 10 minutes in the bath before rinsing in running tap-water. The sections were washed once with Tris-buffered saline (TBS) before being ringed using a resin pen. Either the p53 mouse monoclonal or HSP 70 primary antibody was then added as required and incubated for 1 hour at room temperature. The sections were washed three times with TBS and then biotinylated rabbit anti-mouse secondary antibody added and incubated for 1 hour at room temperature. The sections were again washed three times with TBS and then avidin-biotin complex/horseradish peroxidase added and incubated for 1 hour at room temperature. The sections were washed twice with TBS and then once with Tris buffer (TB) before DAB peroxidase substrate (distilled water 5 ml, pH 7.5 buffer 2 drops, DAB 4 drops and hydrogen peroxide 2 drops) added as reconpnended by the

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manufacturer for 10 minutes. The sections were washed once with TBS and then rinsed in running tap-water before being counterstained with haematoxylin alone. The sections were then dehydrated through increasing alcohol concentrations, cleared in xylene and mounted with a coverslip. Positive p53 protein or HSP 70 protein expression resulted in brown nuclei, negative nuclei appear blue from the haematoxylin counterstain.

Heat Shock Protein 70 (HSP 70) Detection in Mouse Tissue

The method used for detecting HSP 70 expression in the cells o f sections taken from mouse biopsies required a different protocol from that outlined above and was as recommended by LabVision® in the instructions obtained with their Ultravision Mouse Tissue Detection System Anti-Mouse, HRP/DAB.

The sections were incubated, after de-waxing and rehydrating, in the hydrogen peroxide block (kit component) for 15 minutes and then washed twice in TBS. They were then microwaved in 10 mM citric acid buffered to pH 6 as above and left to stand for 10 minutes. The sections were incubated with the Ultra V block (kit component) for 5 minutes at room temperature and then rinsed once with TBS. Incubation with the Rodent block (kit component) occurred for 1 hour at room temperature before rinsing in TBS. HSP 70 primary antibody was added and the sections left for 1 hour at room temperature before rinsing with TBS. The sections were incubated with the Biotinylated goat anti-mouse (kit component) for 15 minutes at room temperature and then again rinsed in TBS. Streptavidin peroxidase (kit component) was applied and the sections incubated for 10 to 15 minutes and rinsed in TBS. DAB chromogen (1 to 2 drops - kit component) was added to 1 ml of DAB substrate (kit component), mixed and applied to the sections for incubation for 5 to 15 minutes before the sections were washed once with TBS. Counterstaining was undertaken with haematoxylin before the sections were dehydrated with alcohol, cleared in xylene and mounted with a coverslip. The positive nuclei appear brown whilst the negative nuclei are counterstained blue.