UA Biophysics:Protocols:Lymphocytes immunostaining with anti-AlphaTubulin

GENERAL GUIDELINES

1. Antibodies are very sensitive to contamination, light, temperature, and dilution. Manage them carefully only in the laminar flow chamber with sterile tips. Do not expose secondary antibodies to light; primary antibodies are less but also photosensitive. Put back antibodies into the fridge as soon as you dilute them. Do not aliquot unless otherwise stated by the manufacturer; in such case, follow their instructions.
2. Once the immunostaining process has begun, the samples may never go dry! Therefore the whole process must be carried out in a wet chamber. This also implies that everything must be ready to be applied in the precise moment.
3. Different antibodies work differently. This protocol might have to be adapted to work with other antibodies. Fixation methods do vary greatly depending on what one wants to label. Optimal concentrations of antibodies also change significantly from between antibodies, between samples or between imaging techniques. Whenever you change one of these parameters you will have to find the optimal working concentration by trial. It is recommended to adjust only one variable at the time, so performing an experiment in which you change both antibodie’s concentrations and the sample, for example, will most likely be deceiving.

OBJECTIVE

The objective of this protocol is to stain microtubules for fluorescence microscopy in human lymphocytes. Cells's nuclei are also stained.

MATERIALS

Materials:

1. Slides with lymphocytes fixed with acetone-methanol 1:1 at -20°C
2. TritonX-100
3. PBS
4. Bovine Serum, Albumine.
5. Primary antibody: Monoclonal mouse anti-alphaTubulin T5168 (Sigma)
6. Secondary antibody: Polyclonal goat anti-mouse IgG Alexa 488 (H+L) A11001 (lifetechnologies)
7. DAPI 300uM
8. 2-Mercaptoethanol 14M
9. Wet chamber
10. Nail polish

RECIPE

Day 1

Have ready:

1. Wet chamber. A wet chamber is simply a Petri dish covered in aluminum foil as to block all light and with a wet paper towel to keep the inside humid.
2. PBS 1x.
3. TritonX-100 0.25%, BSA 1% in PBS (Cell membrane permeabilizating solution).
4. BSA 1% in PBS (Blocking solution).
5. Calculations for desired volume of primary antibody. For immunostaining cells, this primary antibody works appropriately after being diluted 1000x in PBS or in blocking solution.
6. Sterile Eppendorf for antibody dilution.

Procedure:

1. Add 100ul of TritonX-100 0.25%, BSA 1% in PBS to each slide.
2. Incubate in wet camber for 5min at 4°C.
3. Suction + (rinse with several droplets of PBS + suction)x2 times.
4. Have ready solution of primary antibody in blocking solution PBS-BSA 1%. It should not be ready more than 5min before application on slides.
5. Apply 100ul of primary antibody in PBS-BSA 1%.
6. Incubate in humid chamber at 4°C overnight (16 hours in this case).

Day 2

Have ready

1. PBS 1x.
2. BSA 1% in PBS (Blocking solution).
3. Calculations for desired volume of secondary antibody. For immunostaining cells, this primary antibody works appropriately after being diluted 1000x in PBS or in blocking solution.
4. Sterile Eppendorf for antibody dilution
5. DAPI 300uM.
6. 2-Mercaptoethanol 14M.
7. Nail polish.

Procedure

1. Dilute secondary antibody in PBS.
2. Suction + (rinse with PBS + suction) x 5.
3. Apply 100ul of secondary antibody in PBS to each slide.
4. Incubate in humid chamber for 25 mins at 4°C.
5. Apply 1ul of DAPI 300uM to each slide.
6. Incubate in humid chamber for 5 mins at 4°C.
7. Suction + (rinse with PBS + suction) x 5.
8. Let dry.
9. Dilute 2-Mercaptoethanol 14M in PBS to achieve 14mM.
10. Apply 20ul 2-Mercaptoethanol 14mM.
11. Put slide on top.
12. Seal with nail polish.
13. Let dry. Do not expose to light. Ready to be imaged.