Microscopy - Protocols

Sample Protocols
There are many established protocols for fluorescence staining and imaging. We have placed a few here and have listed some web links that can provide more information. Please feel free to consult with Dr. Feramisco, as he may be able to help you choose the most suitable one for your particular needs.

Fluorescent staining of cells:

1. Culture cells on acid washed glass coverslips (Fisher).

2. Fix the cells. The following are some fixation methods in order of preference:

3.7% formaldehyde in PBS for 5' (stock can be stored for 1-2 months)
2.5% paraformaldehyde in PBS 5'
95% EtOH/5% acetic acid 20'
-20C methanol
-20C acetone
10% formalin in PBS 10'

At this point you can wash the cells in PBS and store at 4C overnight or for several days. Staining may be stronger if you proceed directly (dependent on your antibody) with staining.

-Wash 3x with 1x PBS 5 min each (room temp)
-Permeabilize cells with 0.3% Tx-100 in 1xPBS-10 min.
-Wash 3x with 1xPBS-1 min each
-Block with 20% Donkey Serum in 1x PBS( if your secondaries are diluted in goat then block with goat serum…basically whichever animal your secondary is made in, block with that animal's serum in 1x PBS)- 1 Hr at room temp in humidified chamber
-Wash 1x with 1x PBS-5 min
-Incubate with primary antibody: Dilution depends on manufacture recommendations. -Dilute in 20mg/ml BSA in 1x PBS
-Wash 3x with 1x PBS-5 min each
-Incubate with secondary antibody: Dilution depends on manufacture recommendations. Dilute in 20mg/ml BSA in 1x PBS
-Wash 3x with 1x PBS- 5min each
-Incubate with Hoechst (Sigma)- 1:1000 in PBS for 20min at room temp in humidified chamber- This is recommended for nuclear stain. It always helps to have a nuclear stain in case the other antibodies don't work, so you can see if you cells are still there.
-Wash 4x with 1x PBS- 5 min each.
-Dip in double distilled H20 to wash off PBS.
-Mount in gelvatol by putting 4ul on the slide and placing the coverslip on the drop. Do not push down on the coverslip as this can cause it to slide and damage the cells. In the case of live cells or cells that need to stay in a certain medium, seal with cytoseal or valap.
-Let air dry

If staining appears weak try altering incubation time of the primary antibody and dilution of primary antibody.

NOTE: The use of nailpolish as a sealant is discouraged as it can seep under the coverslip and damage the cells thereby preventing optimal images.

NOTE: After mounting, fluorescent specimens should be stored in the refrigerator and protected from light to maximize the lifetime of the fluorescent probes.

Fluorescent Staining of Paraffin Embedded Tissue:

Breast Cancer Breast Tumor imaged by deconvolution microscopy by Monika Szeszel of Dr. Linda Wassernan's lab showing multiplex immunostaining with primary antibodies to estrogen receptor (green), insulin growth factor receptor (red), and nuclei (blue). The volume view was made by Volume Explorer written by Dr. Mike Bailey at the SDSC.

-Deparaffinize tissue
-Perform antigen retrieval if suggested by antibody data sheet. Recommend DAKO 10x Antigen Retrieval Solution
-In order to quench endogenous activity tissue can be treated with 2% sodium borohydride in 1x PBS for 30 minutes and then .1M glycine in DIH20 for 30 minutes.
-Wash 1x with 1x PBS-5min
-Block with 20% Donkey Serum in 1x PBS( if your secondaries are made in goat then block with goat serum…basically whichever animal your secondary is made in, block with that animal's serum in 1x PBS)- 1 Hr at room temp in humidified chamber
-Wash 1x with 1x PBS-5 min
- Incubate with primary antibody: Dilution depends on manufacture recommendations. -Dilute in 20mg/ml BSA in 1x PBS
-Wash 3x with 1x PBS-5 min each
-Incubate with secondary antibody in 20mg/mL of BSA in 1x PBS in humidified chamber at room temp for 1hr.
-Wash 3x with 1x PBS- 5min each
-Incubate with Hoechst 1:1000 in PBS for 20min at room temp in humidified chamber
-Wash 4x with 1x PBS- 5 min each.
-Wash1x with double distilled H20-10 sec.
-Mount in gelvatol with #1.5 coverslips.
-Let air dry

If staining appears weak try altering incubation time of the primary antibody, dilution of primary antibody, antigen retrieval time and solution and different methods of quenching endogenous activity.

Gelvatol Mounting Medium Receipe:

Gelvatol, CelVol trade names for polyvinyl alcohol
(Celanese Chemicals):
Avoid contact with eyes.
Avoid creating dust, as it can form explosive dust/ air mixtures.
Wear gloves and protective eye wear.

1. Add 24.0 gm Celvol/205 to 60.0 gm. glycerol, stir. (use weighing balance)
2. Add 60.0 ml. dH2O and leave for several hours at room temp. In fact, it may take a couple of days to dissolve the Gelvatol.
Notes:
This should be done in a pyrex beaker so it can be gently heated: it should be large enough to accommodate the final volume of solution, since the viscosity makes it unweildly to transfer in mid- preparation.
Cover tightly so that sample does not evaporate.
3. Add 120.0 ml of 0.2M Tris-Cl pH 8.5.
4. Heat, to no more than 50(C with occasional stirring, for a period not to exceed 10 min.
5. When most of the Gelvatol dissolves, clarify by centrifugation at 5000 g X 15 min.
6. Aliquot, air-tight, into 15 ml conical centrifuge tubes and store at 4(C; tubes were filled to capacity, to minimize the air head, and taped shut. Working preps may be left at room temp.

Comment:
Aggressive heating to get polyvinyl alcohol into solution will be evident after the preparation is removed from storage at 4C; the prep will solidify, and must be discarded.

Valap Coverslip Sealant Receipe:

Useful site for an introduction to fluorescent staining:
http://www.itg.uiuc.edu/publications/techreports/99-006/