Immunostaining

2 . 4 . 1 M aterials and Solutions

Paraformaldehyde solution

The stock solution of 20% paraformaldehyde solution was prepared by adding paraformaldehyde powder (BDH) to PBS pre-warmed to 60°C and stirred in a fume hood

until the solution appeared completely clear. Aliquots were stored at -20°C. 4%

paraformaldehyde solution was used to fix tissues and cells. The frozen solution was thawed completely at 37°C and then filtered through a 0.22pm filter before diluting to 3% with PBSABC.

Blocking solution

This comprised 0.2% fish skin gelatin (FSG, Sigma) solution in PBS.

Gelvatol mounting solution

The Gelvatol mounting solution was prepared as described below. 2.4g gelvatol (Monsanto Chemicals) was mixed with 6g glycerol (Sigma) and vortexed. 6ml dH^O was added, mixed and left to stand for 90 minutes at room temperature. 12.5ml of 200mM Tris-HCl, pH 8.5 was added and the solution was vortexed, heated to 50°C and vortexed again.

Heating and vortexing were repeated thrice and the solution placed on a . •

mixer overnight at room temperature, DABCO (l,4-Diazabicyclo-[2,2,2] octane) was added to 2.5% as an antifade agent. The solution was then centrifuged at 750x g for 10 minutes at room temperature and stored in aliquots at 4°C.

Dispose solution fo r removal o f epidermis from dermis

50mg/ml dispase II (Bohringer Mannheim) in DMEM buffered with 20mM HEPES

PE bujfer - blocking and permeabilistion buffer fo r whole mount epidermis staining

0.5% w/v skim milk powder, 0.25% v/v fish skin gelatin (Sigma), 0.5% v/v Triton-xlOO 0.9% w/v NaCl in 20 mM HEPES pH 7.2.

2 . 4 . 2 A ntibodies

Primary Antibodies

Chapter 2 Materials and Methods

Antibody S p ecificity S p ecies R eference

P5D2 human pi integrin mouse monoclonal (Dittel et a l, 1993)

CD29 human (31 integrin mouse monoclonal (Koenigsmann <3/. 1992)

C-FABP E-FABP rabbit polyclonal (Watanabe e ta l, 1996)

LHM2 MSCP mouse monoclonal (Kupsch e t a l , 1995)

3E1 GFP mouse monoclonal (S. Geley - Unpublished)

UCHT4 human CDS mouse monoclonal (Beverley et a l, 1980)

1H9 human MRP-14 mouse monoclonal (N. Hogg - Unpublished)

anti p8 human MRP-8 rabbit polyclonal (Robinson and Hogg., 2000)

anti p l4 human MRP-14 rabbit polyclonal (Robinson and Hogg., 2000)

AC-40 human beta-actin mouse monoclonal (Herman et a l, 1993)

SY5 human involucrin mouse monoclonal (Hudson et a l, 1992)

BC-1 BrdU mouse monoclonal (Campana et a l, 1988)

Secondary Antibodies

Antigen Conjugate S p ecies Source

Mouse IgG Alexa 488 Goat Molecular Probes

Mouse IgG Alexa 594 Goat Molecular Probes

Mouse IgG Phycoerythrin Goat Sigma

Mouse IgG Avidin Rabbit Dako

Mouse IgG HRP Sheep Amersham

Rabbit IgG Alexa 488 Goat Molecular Probes

Rabbit IgG Alexa 594 Goat Molecular Probes

Rabbit IgG Phycoerythrin Goat Moleular Probes

Rabbit IgG HRP Donkey Jackson Immunoresearch

Strepavidin FITC Vector Lab

2 . 4 . 3 Immunofluorescence Staining of Frozen Sections

The Histopathology Unit (ICRF) performed the cutting of all sections.

Frozen sections were dried for 15 minutes then fixed either by a brief dip in ice cold acetone, or in a 3.7% formaldehyde solution in PBS for 5-10 minutes. They were subsequently blocked for 30 minutes in the blocking solution. The sections were washed three times in PBS. Primary antibodies were diluted in the blocking solution and applied to cells for 1 hour at room temperature. The sections were rinsed three times in PBS and then incubated with the secondary antibody, diluted 1:250 - 1-400 in the blocking solution for 30 minutes to Ihour at room temperature. The sections were then rinsed thrice in PBS and once in dH^O before mounting in the Gelvatol solution.

In co-localisation experiments using a double immunofluorescence method, sections were stained with two primary antibodies. Both primary antibodies were of distinct species and these were subsequently detected with species-specific secondary antibodies conjugated to different fluorophore. In this case the order was not impiortnant. Alternatively, one of the primary antibodies was directly conjugated to a fluorophore. In this case, the directly-

Chapter 2 Materials and Methods

conjugated antibody was applied to the cells after incubation with the other primary antibody followed by secondary antibody conjugated to another fluorophore. Stained samples were viewed with a Zeiss Axiophot microscope (Carl Zeiss) or with a Nikon Diaphot 200 inverted microscope linked to an MRC-1000 laser scanning confocal microscope attachment (Bio-Rad).

2 . 4 . 4 Immunohistochemical Staining of Paraffin Sections

Paraffin sections were dewaxed by two five minute incubations in xylene and were then rehydrated by sequential two minute incubations in 100%, 95%, 90%, 80%,70% and 50% ethanol in PBS, followed by two changes of PBS each of 5 minutes. Antibodies were applied as described above. The secondary antibody was peroxidase conjugated. Therefore the final rehydration was adapted to allow for a peroxidase blocking step. After passing through the ethanol series, the sections were incubated for 5 minutes in methanol containing a 10% volume of 30% hydrogen peroxide. Incubation of the secondary antibody was followed by a colour development step in which the sections were incubated in a solution of diaminobenzidine (DAB, Sigma), 0.5 mg/ml with 2.0 pi of hydrogen peroxide per ml. After five minutes, the sections were washed under running water, then lightly counterstained with 10% Geimsa stain in PBS and mounted in Gelvatol as above.

2 . 4 . 5 Immunofluorescence Staining of Cultured Kératinocytes

Cells were cultured on glass coverslips (Chance Propper Ltd.). Coverslips were first

boiled in 7x detergent (ICN) for 30 minutes. They were rinsed thoroughly first in tap water and then in dH^O. Washed coverslips were rinsed briefly in absolute ethanol and spread out on filter paper to dry completely before autoclaving. Cells were routinely fixed and permeabilised. Culture medium was discarded and after 3 washes in PBS, the cells were incubated in 3.7% formaldehyde and 0.4% Triton X-100 in PBS for 5 minutes at room temperature. If there was no need for the cells to be permeabilised, i.e. only detecting surface epitopes, then the Triton X-100 was omitted. The cells were rinsed thrice in PBS and then blocked in 0.2% FSG solution. Application, incubation of primary and secondary antibodies, washing and mounting and image analysis was the same as with the staining of

frozen sections. Confluent sheets were removed as a sheet in 2.5 mg/ml dispase II and 20mM HEPES in E4, and stained as described for frozen sections.

2 . 4 . 6 BrdU Incorporation and Nuclear Counterstaining

Before the staining, kératinocytes on coverslips were incubated in lOpM BrdU for 1 hr in complete FAD medium at 37°C. The coverslips were washed twice in PBS and then fixed in 3.7% Formaldehyde for 10 minutes. The Coverslips were washed twice again in PBS and permeabilised by incubation in 0.5% TritonX-100/2M hydrochloric acid for 10 minutes. Blocking of highly reactive groups was achieved by incubation in lOOmM

ammonium c^hloride for 15 minutes. Again the coverslips were washed . The

coverslips were blocked as normal. The coverslips were incubated with an anti-Brdu, and the second layer was the appropriate secondary antibody. Added to the secondary antibody dilution was a IpMTOPROIII (Molecular Probes) solution. This allowed the visualisation of all the nuclei. Images were taken by confocal microscope as described in staining of frozen sections.

2 . 4 . 7 Immunostaining of Wholemount Epidermis

Details of the whole mount staining technique and its applications have already been

described (Jensen et a l., 1999). Normal human skin was obtained from cheek and breast.

The fat and most of the connective tissue was removed with a scalpel. The skin was divided into 1-1.5 cm^ pieces and incubated in dispase solution. Using forceps the epidermis was gently removed from the underlying dermis as an intact sheet and fixed immediately in normal buffered formalin pH 7.2, for 2 hours at room temperature. Epidermal sheets were stored in PBS containing 0.2% sodium azide for up to 8 weeks prior to staining. Epidermal sheets were simultaneously permeabilised and blocked in PB buffer for 30 minutes. The primary antibodies were diluted in PB buffer and incubated at room temperature overnight with gentle agitation. The epidermis was then washed in PBS containing 0.2% Tween-20 for 3-4 hours with several changes of wash buffer. Incubation in secondary antibodies was performed in the same way. The epidermal sheets were mounted dermal papillae upwards in an excess of Gelvatol.

Chapter 2 Materials and Methods

Mounted wholemounts were analysed with a Nixon Diaphot 200, as with the sections and the fixed cells. Horizontal sections of 0.5 or 1pm thickness were taken through the wholemount from the just beneath the rete ridges to just above the dermal papillae, a thickness of around 60ju\m. These sections could be analysed individually, or a projection of all the sections could be produced. These projections could be re-coloured with a false colour representation of the intensity of the signal.