Respuestas (Códigos de estado) SIP.

4.3.1 Preparation of cultured cells for immunocytochcmical staining

Adherent cells were grown on high grade microscope coverslips (Chance Proper Ltd., Warley, UK) according to the method of Harlow and Lane (1988). Coverslips were sterilised by dipping in 70% v/v ethanol and flaming, then they were aseptically transferred to petri dishes under a flow hood. Cells were seeded into the petri dishes at a density of 105 ml'1, transferred to a humidified incubator at 37°C, 5% C 0 2 and allowed to attach to the coverslips for 24 hours. For nonadherent or partially adherent cells such as PC 12 cells, the coverslips were treated with poly-L-lysine and sterilised by overnight exposure to UV light under a flow hood prior to seeding. Cells were fixed by rinsing the coverslips in PBS and incubating in 4% w/v paraformaldehyde (pH 8.0) in PBS for 10 minutes at room temperature. The cells were then washed twice with PBS and permeabilised by dipping into 0.2% v/v Triton X-100 in PBS for 2 minutes. Prior to staining with antibodies, the coverslips were washed in PBS, 3x 5 minutes.

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4.3.2 Preparation of mouse embryos and adult tissue for immunohistological staining

Pregnant female mice were sacrificed by cervical dislocation and embryos were dissected into cold PBS. After rinsing, litters were transferred to 20ml Sterilin tubes and fixed in 4% w/v paraformaldehyde/PBS (pH 8.0) on ice. The fixation time was varied from 30 minutes to 12 hours according to the size of the specimens and embryos more advanced than E12.5 were pierced to facilitate penetration o f the fixative. After fixation, litters were transferred to fresh 20ml Sterilin tubes and equilibrated first in 5% then 15% w/v sucrose/PBS on ice. Meanwhile, a solution of 7% gelatin, 300 Bloom (Sigma Chemical Co., St. Louis, USA.) in 15% w/v

sucrose/PBS was prepared by boiling in a microwave oven and cooling to 37°C in a waterbath. The embryos were transferred to fresh 20ml Sterilin tubes containing

10ml gelatin solution at 37°C and incubated for 30 minutes. They were then

transferred to plastic moulds, oriented and embedded in gelatin overnight at 4°C. The blocks were trimmed into trapezoid shapes to emphasise orientation and frozen by immersion in isopentane over liquid nitrogen. They were stored in Sterilin tubes at -70°C prior to sectioning. Adult tissues were dissected, cut into blocks approximately 5mm3 if necessary and prepared in the same manner as embryos.

4.3.3 Sectioning of mouse tissue

Blocks stored at -70°C were transferred to the cryostat cabinet at -20°C, mounted onto the chuck using Tissue-Tek O.C.T. compound (Miles Inc.) and trimmed. Sections were cut at 10pm and collected onto gelatin-subbed slides stored at room temperature.

10-20 sections were collected onto each slide. They were briefly air dried and used immediately for immunohistological staining.

4.3.4 Immunological staining - general strategy

Specimens subjected to immunological staining for specific proteins were also subjected to control reactions in order to verify the results. As a negative control, specimens were incubated in blocking agent whilst the experimental specimens were exposed to the primary antiserum. Both the experimental and control specimens were then exposed to the secondary antiserum. This strategy controlled for nonspecific

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binding o f the secondary antiserum and autofluorescence. As a positive control, a number o f antisera known to bind to specific tissues were used. Control specimens were incubated with these antisera whilst the experimental specimens were exposed to their primary antiserum. Both the experimental and control specimens were then exposed to the secondary antiserum. This strategy controlled for failure of primary or secondary binding and allowed nonambiguous identification of specific cell types.

4.3.5 Immunocytochemical staining of cells

Cell staining was carried out according to the method o f Harlow and Lane (1988), pp 392-393. Coverslips were transferred to a damp chamber lined with water repelling film (Nescofilm) and blocked with 3% w/v BSA/PBS. Primary antisera were used at manufacturers' recommended working dilutions and coverslips were incubated in the foil-wrapped chamber for 3 hours. The coverslips were then rinsed in three changes of 100ml PBS containing 1% v/v Triton X-100, blotted on a tissue to remove excess liquid and incubated with the secondary, FITC-conjugated antiserum. The optimal working dilution for the secondary antiserum was determined empirically by titration and incubation was carried out as above. The cells were washed and dried as above then mounted on clean glass slides with 50% v/v glycerol/PBS. No anti-quenching additives were used as the stained sections were observed and photographed immediately, as described in section 3.1.2.

4.3.6 Immunohistochemical staining of tissue sections

Sections were blocked and stained according to the method of Hogan et al. (1986), pp 243-4. Primary antisera were used at the manufacturers' recommended working dilutions, 250pl per slide. Incubation was carried out for 1 hour in a foil-wrapped damp chamber on a tilting platform. Slides were then transferred to a rack which was placed in a staining jar containing a small magnetic stirrer and washed for 5 minutes each in three changes of 400ml PBS. The optimal concentration of the secondary, FITC-conjugated antiserum was determined empirically by titration. Tissue paper was used to dry as much of the slides as possible without disturbing the sections and the secondary antisera were added, 250pl per slide. Incubation was carried out for 30 minutes as above. The slides were washed and dried as above and the sections were mounted in 50% v/v glycerol/PBS. No anti-quenching additives were used in the

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mountant and the stained sections were observed and photographed immediately, as described in section 3.1.2.

4.4 In situ hybridisation techniques

4.4.1 In situ hybridisation - General strategy

Analysis of NSE expression at the mRNA level during mouse development was facilitated by the use of antisense RNA probes corresponding to a distal portion of the rat NSE 3' UTR. The UTRs of the three mammalian enolase genes are highly divergent, allowing isogene-specific probes to be generated (see section 1.6), however, there is strong orthologous conservation o f the UTRs between species allowing heterologous probes to be used with success. NSE mRNA expression was studied by wholemount in situ hybridisation using digoxigenin-UTP-labelled antisense RNA probes synthesised as described in section 4.1.6.3. Sense probes generated from the same construct were used as controls for non-specific

hybridisation. Further controls, where no probe was included in the hybridisation step, were used to detect nonspecific binding of the antibody or accumulation of colouring reagents.

4.4.2 Preparation of embryos

Wholemount in situ hybridisation was carried out according to the procedure described in the following sections. This was optimised by Dr D Stott from standard in situ hybridisation protocols (Wilkinson, 1992). Embryos were dissected into cold PBS and the extraembryonic membranes were removed with fine forceps. The embryos were transferred to 20ml Sterilin tubes and fixed on ice in 4% w/v

paraformaldehyde/ PBS (pH 8.0) overnight. All following steps were carried out in microcentrifuge tubes at room temperature for 5 minutes using 1 ml of solution and gentle end over end rotation, unless stated otherwise. Following fixation, the embryos were washed twice in PBS, refixed in 4% w/v paraformaldehyde/PBS (pH 8.0) at 4°C for 20 minutes, washed twice in PBST (1% v/v Tween 20/PBS) and then incubated with proteinase K (20 pg mP1 in PBST). The duration of proteinase K treatment depended upon the size of the specimen: embryos at E9 or earlier were incubated for 3 minutes, those at E9.5-El0.5 for 5 minutes. The embryos were then rinsed briefly in PBST, washed in PBST then postfixed for 10 minutes in 4% w/v

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paraformaldehyde/PBS (pH 8.0). After a brief rinse in distilled water, the embryos were treated for 10 minutes with 0.1 M triethanolamine (pH 8.0) (Analar BDH) containing 2.5 pi mb1 acetic anhydride (Analar BDH), to reduce background staining. The embryos were transferred to glass vials, washed twice in PBST and were then ready for hybridisation. If storage of the embryos was required, they were transferred to glass vials containing 50% v/v ethanol after the first fixation and wash step. They were then equilibrated in several changes of 70% v/v ethanol and stored at 4°C in the dark. Stored embryos were stable for at least three months and were rehydrated by incubating in 50% v/v ethanol, 30% v/v ethanol/PBS followed by two washes in PBST before further processing.

4.4.3 Wholemount in situ hybridisation to mouse embryos

The following steps were carried out at 50°C in a waterbath without rotation. The embryos were equilibrated for 1 hour in hybridisation solution without blocking agents (50% v/v deionised formamide, 5x SSC, 20mAf Tris.Cl (pH 8.0), 5mA/EDTA, 0.1% v/v Tween 20). The hybridisation solution was removed and replaced with fresh, plus an equal volume o f hybridisation solution with blocking agents (50% v/v deionised formamide, 5x SSC, 20mMTris.Cl (pH 8.0), 5mA/EDTA, 0.1% v/v Tween 20, 0.2% w/v polyvynilpyrrolidone, 0.2% w/v Ficoll type 400, 2mg mb1 heparin, 2mg mb' yeast RNA; all blocking agents obtained from Sigma Chemical Co., St. Louis, USA.) and prehybridisation was carried out for at least 2-6 hours. The probe was added to the hybridisation mix to a final concentration o f 0.1 pg mb' and hybridisation was carried out overnight.

4.4.4 Posthybridisation washes

Following hybridisation, the embryos were washed at 50°C for 20 minutes in 50% v/v deionised formamide, 2x SSC, then at 37°C for 3x 10 minutes in NTET (0.5A/NaCl,

lOmAf Tris.Cl (pH 7.5), 5mA/EDTA, 0.1% v/v Tween 20). The embryos were then treated with RNase for 30 minutes at 37°C (final concentrations 20pg mb1 RNase A,

100U mb' RNase T1 in NTET) and washed again in NTET for 10 minutes. The embryos were then washed in 50% v/v deionised formamide, 2x SSC, 50°C for 1 hour; 2x SSC, 0.1% v/v Tween 20, 50°C for 1 hour and 0.2x SSC, 0.1% v/v Tween 20, 50°C for 1 hour. They were then equilibrated with PBST.

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4.4.5 Signal detection

Signal detection was carried out using the DIG detection kit (Boehringer Mannheim, Mannheim, Germany) according to manufacturer's instructions. Briefly, nonspecific binding sites were blocked by incubating the embryos with 1ml 0.5% w/v Boeringher blocking compound in PBST for 1 hour at room temperature. 0.5pl of Boehringer antidigoxigenin antiserum was then added (final concentration 1:2000 dilution from stock) and the reaction was incubated overnight at 4°C. The next day, the embryos were washed in PBST at room temperature, 6x 1 hour. The colour reaction was then set up by washing the embryos twice, for 5 minutes each time, in 1ml colour buffer (O.lA/Tris.Cl (pH 9.5), 50mMMgCl2, O.lAfNaCl, 0.1% v/v Tween 20, 5mM levamisole (Sigma Chemical Co., St. Louis, USA)) and then adding 4.5pl nitroblue tetrazolium and 3.5pl X-phosphate (5-bomo-4-chloro-3-indolyl

phosphate) from the kit. The reaction was left to colour for 24 hours, then the embryos were cleared in Murray's reagent (50% v/v benzyl alcohol/benzyl benzoate) and photographed as described in section 3.1.2.