ENSAMBLE DEL EQUIPO DE ESTEREOLITOGRAFÍA SLA

2.5.1 Preparation of tissue cryosections

Principle: Tissue samples (pre-fixed or not) are frozen, cut into thin sections using a cryostat, mounted on glass microscope slides and stored at -70°C indefinitely.

Materials:

- glass microscope slides;

- industrial methylated spirits (IMS);

- 2% 3-aminopropyltriethoxysilane (Sigma) in IMS; - 4% paraformaldehyde in PBS;

- 30% sucrose in PBS;

- OCT tissue freezing compound (Tissue Tek).

Preparation of glass microscope slides:

1. Place the slides in plastic racks and immerse in IMS for 5 minutes.

2. Transfer the racks in 2% 3 -aminopropyltriethoxysilane (Sigma) in IMS and incubate 5 minutes.

3. Wash the slides twice in deionised water, then briefly dehydrate in IMS. Place them on a paper towel to dry, for 60 minutes, then bake at 65°C for two hours. Store in an air-tight box.

Preparation of tissue samples and sectioning:

1. Tissue samples are either frozen immediately after surgical decapitation or fixed overnight in 4% paraformaldehyde in PBS and cryoprotected in 30% sucrose in PBS for another 24 hours.

2. Place the tissue in a mould with OCT compound and orient it in the desired position. Then transfer it on a dry ice/pentane bath until frozen. Store at -70°C.

3. For section, warm the frozen tissue pieces to -20°C and cut 10-12 pm thick sections in a cryostat (Bright Instrument Company).

4. Place the freshly-cut sections on a glass slide, let them air-dry, incubate 2 hours at 37°C and then wrap the slides in aluminium foil and store at -70°C.

2.5.2 Immunohistochemistry and immunocytochemistry

Principle: Antibody detection of specific antigens exposed in tissue sections or expressed by adherent cells. This example illustrates the use o f an unlabelled primary antibody and fluorochrome-labelled secondary antibody. When horseradish peroxidase- labelled secondary antibodies were used, they were localised using diaminobenzidine and hydrogen peroxide as substrates.

Materials:

- tissue sections mounted on glass microscope slides OR adherent cells grown on extracellular matrix components-coated glass coverslips;

- humidified plastic chamber for incubations;

- pre-block solution: 1% w/v bovine serum albumin (BSA), 0.2% Triton X-100 in PBS; - antibody dilution solution: 3% w/v BSA, 0.05% Triton X-100 in PBS;

- wash solution: 0.1% w/v BSA, 0.05% Triton X-100 in PBS; - antibodies (unlabelled primary, fluorochrome-labelled secondary); - mounting reagent: Fluorosave (Calbiochem).

1. Rinse the slides/coverslips briefly in PBS.

2. Fix the tissue sections/cells either with 4% paraformaldehyde, 5 minutes at room temperature or with ice-cold absolute methanol, 1-5 minutes at -20°C (The fixation method depends on the antigen sensitivity).

3. Rinse the slides/coverslips briefly in PBS, twice. If using microscope slides, draw around the sections of interest a hydrophobic circle using a wax pen, to prevent leakage of antibody solutions.

4. Block non-specific binding sites by covering the tissue sections/cells with pre-block solution. Incubate 15 minutes at room temperature.

5. Remove the pre-block solution and incubate the tissue sections/cells with the primary antibody, adjusted to the working concentration with dilution solution. Incubate 1 hour at room temperature or overnight at 4°C.

6. Wash the slides/coverslips in wash solution 3 times, 5 minutes each, with shaking, at room temperature.

7. Incubate the tissue sections/cells with the secondary (labelled) antibody adjusted to the working concentration with dilution solution. Incubate 1 hour at room

temperature. At this step other staining reagents (e.g. phalloidin-TRITC) can be added.

8. Repeat step 6.

10. Mount them in Fluorosave, allow the reagent to solidify 1 hour at room temperature and then store the slides up to two weeks, in dark, at 4°C.

2.5.3 Receptor Affinity Probe (RAP) in situ

Principle: Cell-surface receptors usually bind their ligands with high affinity and specificity. Therefore, soluble receptor ectodomains, usually tagged at their carboxy terminus, can be used as probes to detect their ligands in various assay formats. The heat-stable human placental alkaline phosphatase (AP) is commonly being used as a C terminus tag (Flanagan et al., 2000). It has the advantage of possessing an intrinsic enzymatic activity that allows easy detection and accurate quantification.

Materials: All common chemicals were purchased from Sigma.

- tissue sections mounted on glass microscope slides OR adherent cells grown on extracellular matrix components-coated glass coverslips;

- AP fusion protein: 7-day conditioned medium obtained from 293T cells transiently transfected with a plasmid encoding the construct of interest; this medium is buffered with lOmM HEPES, pH 7, sterile filtered (0.45 pm), and stored at 4°C;

- humidified plastic chamber for incubations;

- fixatives: ice-cold absolute methanol and fresh 4% paraformaldehyde in PBS, pH 7.5; - HBAH buffer: Hanks’ balanced salt solution, 20 mM HEPES, pH 7.0, 0.5 mg/ml

bovine serum albumin, 0.1% w/v NaNg;

- HBS buffer: 10 mM HEPES, pH 7.0, 150 mM NaCl;

- AP buffer: 100 mM Tris.HCl, pH 9.5, 5 mM MgClz, 100 mM NaCl;

- NBT/BCIP (nitroblue tétrazolium / bromochloro-indolyl phosphate) substrate mix (Roche Molecular Biochemicals).

2. (Optional) Fix the tissue sections/cells either with ice-cold absolute methanol, 2 minutes at -20°C or with 4% paraformaldehyde, 5 minutes at room temperature (The fixation method should be tested and optimised for each receptor/ligand pair).

3. Rinse twice, 90 sec each, in HBAH buffer.

4. Add AP fusion protein to cover the tissue sections/cells and incubate 90 minutes at room temperature

5. Wash the slides/coverslips six times, 90 sec each, in cold HBAH buffer. 6. Fix in 4% paraformaldehyde, 90 sec at room temperature.

7. Wash the slides/coverslips twice, 90 sec each, in HBS.

8. Incubate the sections/coverslips in pre-heated HBS, in a 65°C waterbath, for 30 minutes - 1 hour.

9. Wash the slides/coverslips in AP buffer, 5 minutes.

10. Add AP buffer containing NBT/BCIP to cover the tissue sections/cells and incubate at room temperature in a dark humidified plastic chamber. Monitor the reaction

periodically until the desired signal intensity is achieved.

11. Stop the reaction by washing briefly with distilled water and mount the slides/coverslips.