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1. GMA sectioning.
- Prepare 0.05% ammonia solution with distilled water. Place the glass knife and biopsy in the microtome. Carefully align the biopsy block with the blade by adjusting the knife holder and the angle of the biopsy block so that the blade rests flat against the surface of the block.
- Cut 2 µm thick sections using the microtome on a slow cutting speed and use forceps to transfer and float out sections on the ammonia water. Float the sections for 45-90 sec, allowing gentle antigen retrieval and unfolding of the section.
- Pick up sections with microscope slide. Check the biopsy histology by quickly staining with toluidine blue.
- Dry the slides on a hot plate set to 50 °C and apply 500 µl of filtered toluidine blue stain to a section using a plastic Pasteur pipette. Warm the section on the hot plate until a green ring begins to emerge at the edge of the stain, and then wash it off with water.
- Using a light microscope, check the biopsy histology. Sections used for immunohistochemistry should contain good areas of undamaged lamina propria and epithelium, with as few glands and as little smooth muscle as possible.
- Cut sections that have good histology and pick them up with poly L-lysine-coated (PLL-coated) microscope slides for immunohistochemical staining. Cut at least two sections from each biopsy for analysis. Dry slides for at least 1 hr. After drying, sections can be wrapped in tin foil and stored at -20 °C for up to 2 weeks, or they can be immediately immunostained.
2. Immunohistochemical Staining of GMA-embedded Endobronchial Biopsies
NOTE: Sodium azide is toxic. Prepare the 0.1% sodium azide solution within a fume hood and away from acids. Contact with acids liberates toxic gas.
- Draw around sections using a diamond-tipped pen and arrange the slides onto a staining rack.
- Perform a Peroxidase block. Prepare a peroxidase block solution of 0.3% hydrogen peroxide in 0.1% sodium azide solution. Apply 1 ml of the peroxidase block to sections and incubate for 30 min.
- Prepare a wash solution of 0.05 M Tris-buffered saline (TBS), pH 7.6. Wash the slides in TBS, 3x 5 min.
- Prepare a 1% BSA block solution in DMEM. Do this in advance and in large volumes, aliquot and freeze it until needed. Drain the slides and incubate the sections in block solution for 30 min to block unspecific antibody binding. If unspecific binding still occurs, include a 30 min incubation step with 5% serum block from the same species as the secondary antibody.
- Dilute primary antibody (CD45 or CD1a) in TBS to the desired concentration (CD45 diluted at 1:1,000; CD1a diluted at 1:1,00) and apply it to slides. Coverslip the sections and incubate overnight at RT.
- Wash slides in TBS three times. Dilute biotinylated secondary antibody (directed against the primary antibody host species, in this case rabbit anti-mouse F(ab'2)) in TBS to the desired concentration (1:300 dilution) and apply it to slides. Incubate for 2 hr at RT.
- Prepare a streptavidin biotin-peroxidase complex at least 30 min before use. Ensure that there is enough to cover all slides. Wash the slides in TBS three times. Apply the solution to the slides and incubate for 2 hr at RT.
- Wash the slides in TBS three times. Prepare 3-amino-9-ethylcarbazole (AEC) peroxidase substrate solution (made per the manufacturer's instructions). Apply it to the slides and incubate for 20-30 min or until the desired stain intensity develops.
- Rinse the slides in running tap water for 5 min. Counterstain the sections with filtered Mayer's haematoxylin solution for 2 min. Wash the slides in running tap water for 5 min.
- Drain the slides and cover the sections with permanent aqueous mounting medium. Dry the slides at 80 °C in a drying oven. Allow the slides to cool, and then mount them with DPX and place a coverslip.