Immunostaining of fish scales

WHOLE MOUNT IHC ON ZEBRAFISH SCALES

(Adapted from Iyengar et al. 2012 J. Vis. Exp.)

Important note: the exposed part of the zebrafish scale contains the epidermis and therefore, the majority of the cells, vessels, etc. so be gentle when plucking them!

Day 1:

1. Anesthetize the fish in tricaine (following lab’s protocol).

2. Pluck scales using sharp fine-tipped forceps taking care not to damage the scale. If necessary, sample them under a fluorescence dissecting microscope.

3. In a 2ml tube (round bottom), put the scales in 1 ml at a final concentration of 10mM DTT/PBS, 1h at RT (agitation)

4. Wash 2 x 5’ in PBS (agitation).
Careful to not loose scales! They are very difficult to see unless they have some pigmentation.

5. Fix in 4% PFA pH 7.4 at RT 1h (agitation).

6. Transfer the tube on ice for 3h (flip from time to time).

7. Wash 3 x 10’ in PBS-Tx0.1% (agitation). Careful to not loose scales!

8. Blocking: at least 2h incubation at RT using the blocking buffer specified below (it has a different composition from the one normally used in the lab). Use secondary antibody species serum. Careful to not loose scales!

9. Primary antibody incubation O/N at 4ºC under agitation diluted in blocking buffer (200-300 µl/well). Careful to not loose scales!

Note: if performing control negatives, incubate with blocking buffer only.

Note: I have also tested Ab1º incubation O/N at RT under agitation and it also worked (as specified in the original paper) but this also it may depend on the stability of the Ab1 used.

Day 2:

1. Quick wash with PBS-0.1% Tx. Careful to not loose scales!

2. Wash and 3 x 10’ PBS-0.1% Tx (agitation). Careful to not loose scales!

3. Secondary antibody incubation + DAPI (1:2000): 3h at RT in under agitation diluted in blocking buffer (200-300 µl/well). ALU FOIL! KEEP PROTECTED FROM LIGHT.

4. Wash well with PBS-0.1% Tx and 3 x 10’ (agitation).
Careful to not loose scales! KEEP PROTECTED FROM LIGHT.
Note: this washing step will ensure that there is no remaining Ab2 that may give background or little fluorescent spots on the scales. Scales are sticky so in the immuno often there is “dust” signal.

5. Mounting is a tricky step. It takes time to catch the scales with the forceps without damaging them.

• Warm the Glycergel (DAKO) at 50°C.

• Under the dissection microscope, take the scales from the tube, one by one if possible and put them on a slide. I put them in different rows and I pipet the excess of liquid before adding the Glycergel. DO NOT LET THE SCALES TO DRY.

Tip: I pipet the scales from the tube with a P1000 cut and I put them on a Petri dish so it is easier to take them with the forceps.

It is possible to do it under a fluorescence microscope but then try to keep them wet and go to the lab to add the Glycergel. In the original paper they recommend to place them with the concave side of the scale facing down towards the slide.

• Put a drop of Glycergel (DAKO) carefully on each scale and add more glycergel around the slide to make sure it will spread.

• Place a cover glass and push softly. Do not press too much (scales are thick) and eliminate any bubbles. Store the slides at 4°C. KEEP PROTECTED FROM LIGHT. Check if the immune worked under a fluorescence dissecting microscope.

SOLUTIONS

DTT diluted in PBS (stock at 100mM) --> 100ul DTT 100mM in 900ul PBS = 1ml

Blocking buffer (2mg/ml BSA, 5% serum, 0.2% Triton, 5% serum)

PBSTx (0,1% Triton X-100) for 50 ml

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