Protocol for fluorescent detection on paraffin embedded samples:

Deparaffinization -

Citrus solvent (used in place of xylene)  3 x 3 min

Ethanol    100%                                   2 x 2 min

                 95%                                    2 x 2 min

                 80%                                    2 x 2 min

                 50%                                    2 x 2 min

Rinse 5 min in flowing water (Barnsteadt or Milli Q)

Retrieval - in citrate buffer pH6, boiling for 9 min (in microwave on high for 3 min, then 6 min with pulse of 10 sec at 50% during every 30 sec to keep it boiling.)

Rinse 3 x 5 min in PBS.

Block:  incubate 1 hr in normal donkey serum diluted 1 to 50 with PBS (2% v/v) at RT

Primary antibody:  CT-1 (peptide affinity purified rabbit polyclonal recognizing the cytoplasmic tail of Muc1) diluted 1 to 10 with PBS.   Incubate for 1 hr at 37C.

Rinse 3 x 5 min with PBS.

Secondary antibody:  FITC conjugated donkey anti- rabbit IgG (Amersham) diluted 1 to 10 with PBS.  DAPI was included with the second antibody to counterstain nuclei.

Incubate 40 min at 37C.

Rinse 3 x 5 min with PBS.

Mount in glycerol based mountant (I make ours) which includes an antifadant.

Note that this protocol is tailored for MUC1 detection with CT-1 antibody.  You may want to add a quenching step if you are using paraformaldehyde fixation - 50 mM ammonium chloride in PBS for 30 min.  It may be used as one of the rinse steps after the retrieval step.  For other epitopes you may have to add an unmasking step - or some form of digestion to aid penetration in some tissues.