Cell Biology Lab protocol

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Immunofluorescent staining of Drosophila melanogaster ovaries for actin and CHICO.

Drosophila oocytes undergo periods of yolk protein uptake via nurse cells followed by rapid cytoplasmic transport via ring canals (see below). YP uptake from the hemolymph into the nurse cells (below) is via receptor mediated endocytosis. The ring canals are formed partly from actin and filamin (below) proteins linked together in structures that reach about 10 micons in size. As we now know, receptor mediated endocytosis involving possible regulation by the insulin-signaling pathway is a part of the process of vitellogenesis

Photograph of ring canals in developing oocyte from the Lynn Cooley website at Yale.

Filamin (green) and actin (red) in complex.

We will stain for CHICO using an primary antibody raised in rabbit against a 20-amino acid fragment of the CHICO protein (predicted from the cDNA sequence). This will then be probed with a goat anti-rabbit IgG secondary antibody coupled to the red fluorescent label rhodamine (TRITC). We will stain for actin using an actin-binding fungal toxin called phalloidin which has been conjugated to the fluorescent label fluoroscein (FITC). Then using different wavelengths of stimulating light we can observe both labels simultaneously under laser confocal miscrocopy (hopefully!) In case that doesn't all work (which is possible as we are doing this over a 2-week period), we will also set up a series of ovaries just stained for actin using the phalloidin-FITC conjugate.

Yolk protein receptor localization to nurse cells adjacent to the developing oocyte (Richard et al., 2001)

Day 1: Tuesday 14th November:

Fixation of ovaries.

• Dissect out ovaries in cold EBR and transfer to 1.5ml microfuge tube containing cold EBR on ice.Ovaries will be dissected prior to the lab and stored at 4^oC in EBR
• Remove EBR with Pasteur pipette. Add 100ul of devitellinizing buffer and 600ul heptane. Agitate vigorously to ensure buffer is saturated with heptane and then incubate gently for 10 minutes at room temperature.
• Remove solution with a Pasteur pipette and rinse with PBS three times, and then incubate with 100ulPBS 3x for 10 minutes each.

Antibody staining in ovaries

• Gently tease apart the ovaries using needles
• Incubate in 100ul PBST (not the same as PBS, it has the detergent TritonX-100 added) for 10 minutes.
• Add 3ul anti-CHICO antibody (developed in rabbit) to tube and incubate at 4^oC.

Day 2: Tuesday 21st November:

• Rinse with PBST 3x and then wash with 200ul PBST 4x for 10 minutes each.
• Add 2ul TRITC conjugated anti rabbit IgG antibody Incubate at 4^oC until the following week.

Day 3: Tuesday 28th November:

• AM: Add 5ul of phalloidin-FITC conjugate to tubes containing fresh ovaries in the morning, and to the CHICO stained ovaries. (By this stage then you will have one tube stained for CHICO and actin, and a second new tube stained just for actin)
• Rinse with 100ul PBST, then wash with PBST 4x for 10 minutes each. Rinse 2x with PBS
• Add 100ul PBS:Glycerol (1:1) incubate at room temperature for 20 minutes.
• Place ovaries on microscope slide with glass shards under the cover slip and examine using laser confocal/fluorescent microscope at wavelengths for both FITC and TRITC staining.