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Preparation of samples for light/confocal microscopy Source: Multi-parametric bioimaging and cytometry core facility of the OVGU Note: Different specimens may require different preparation methods. Therefore, the testing and optimizing of e

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Preparation of samples for light/confocal microscopy

Source: Multi-parametric bioimaging and cytometry core facility of the OVGU

Note: Different specimens may require different preparation methods. Therefore, the testing and optimizing of each step within the protocol for each new sample type will ensure that the best balance between preservation and labeling is obtained.

This protocol was optimized for fibroblast cells:

Step Temperature Duration

Fixation 4 %PFA/0.02 %GA in PBS (pH 7.4) RT 10 min

wash 3x in PBS RT 5 min

Permeabilization 0.2 % TX100 in PBS RT 5 min

wash 3x in PBS RT 5 min

Blocking-step 1 % BSA in PBS RT 10 min

Primary antibody incubation RT 1 h

wash 3x in PBS RT 5 min

Block 1 % BSA in PBS RT 10 min

Secondary antibody incubation RT 1 h

wash 3x in PBS RT 5 min

Postfixation 4 % PFA/0.05 % GA in PBS RT 10 min

wash 3x PBS RT 5 min

wash 1x PBS (pH 8.9) RT 5 min

Embed in mounting medium:

Vectashield (Vector Labs) + glycerol 1:1 and 2 % DABCO Sealing the samples:

Nail polish or nail hardener or VALAP.

VALAP is a 1:1:1 mixture of vaseline, lanolin and paraffin. It is prepared by mixing the components in a bottle on a heating plate. The mixture is applied with the help of a thin tip and hardens immediately. It can be reheated and reused many times.

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