General Staining Protocol

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in staining samples fixed according to the Paraformaldehyde Fixation protocol.

Necessary Materials

1. Tween20

  • Purchased from Sigma (P9416) and should be stored at room temperature upon arrival.
  • Note: This detergent is labeled as a corrosive and should be stored accordingly.

2. Triton X-100

  • Purchased from Invitrogen (X100) and should be at room temperature upon arrival.
  • Note: This detergent is labeled as a corrosive and should be stored accordingly.

3. Phosphate Buffered Saline Solution with Mg+ and Ca+

  • Purchased from many places. I use Hyclone (SH30264.02) that can be purchased from GE Healthcare Life Sciences.
  • Upon arrival should be stored at 4°C

4. Molecular Biology Grade Water

  • Purchased from Corning (Cellgrow) (46-000) and should be stored at room temperature upon arrival.

5. Blocking Buffer (see blocking buffer section for specific requirements)

  • Typically use:
  • Fetal Bovine Serum purchased from Invitrogen (10437-028) which should be aliquoted and stored at -80°C upon arrival.
  • Goat Serum purchased from Invitrogen (16210072) which should be aliquoted and stored at -80°C upon arrival.

6. Staining Reagents (see list for specific protocols)

7. DAPI

  • Purchased from Sigma (D9542) and should be stored in 4°C upon arrival.

8. Non-tissue culture treated plates (typically use 6 well)

9. Bibulous Paper

10. No. 1.5 Glass Slides

11. Mounting Agent

12. Clear Nail Polish

Procedure

0.05% Tween20 Wash Buffer Preparation

For 500 mL of wash buffer

1. Add 500 mL of PBS[+] to a glass bottle.

2. Add 250 uL of Tween20 into the PBS[+].

  • Note: Tween20 is very viscous and should be transferred slowly.

3. Shake bottle until Tween20 is completely dissolved.

4. Label with: (write on tape not on the glass)

  • Date made:
  • Solution Name: 0.05% Tween20
  • Your name:

5. Store at room temperature.

0.1% Triton X-100 Wash Buffer Preparation

For 500 mL of wash buffer

1. Add 500 mL of PBS[+] to a glass bottle.

2. Add 500 uL of Triton X-100 into the PBS[+].

  • Note: Triton X-100 is very viscous and should be transferred slowly.

3. Shake bottle until Triton X-100 is completely dissolved.

4. Label with: (write on tape not on the glass)

  • Date made:
  • Solution Name: 0.1% Triton X-100
  • Your name:

5. Store at room temperature.

Blocking Buffer Selection and Preparation

Important: If doing antibody staining - blocking buffer host should be selected based on the host of your secondary antibodies.

1. Select the correct serum to use in your blocking buffer preparation.

  • Example: For goat secondary antibodies use goat serum as the blocking serum.

2.

Typical Serum Percentages Used:
Goat Fetal Bovine
5% 10%

For 10 mL of blocking buffer

3. Add (9.5 mL if GS|9 mL if FBS) of PBS[+] to a conical tube.

4. Add (500 uL if GS|1 mL if FBS) of the selected buffer serum.

5. Agitate until mixed.

6. Aliquot into 1 mL tubes and store in -20°C storage.

7. Label tubes with

  • Date made:
  • Solution Name: BB (5% GS|10% FBS) in PBS[+]
  • Your name:

Staining Protocol

1. Follow the appropriate staining protocol:

Important: Work in a dark room whenever handling uncovered fluorescent stains.

2. Once samples have been stained with the appropriate markers rinse briefly in DI water.

3. Dry samples on a clean sheet of bibulous paper and place cell side up onto the paper.

4. Pipette ~10 uL of anti-fade mounting solution onto the top of the sample.

  • Take care not to have any bubbles in the mounting solution!

5. Using a No. 1.5 glass slide (or appropriate thickness for your microscope) gently lower the slide over the sample.

6. After sample is mounted label with:

  • Stain Used:
  • Sample Type:
  • Culture Used:
  • Date Stained:
  • Your name:

7. Place into a storage at room temperature for ~4 hours.

8. Seal the outside edges of the sample to the slide using clear nail polish.

9. Store at 4°C.

10. Pipette ~80 uL of DAPI staining solution onto each sample (enough to fill only the top of the sample).

11. Incubate with DAPI staining solution for 5 minutes.

Cleaning Up

1. Dispose of the used buffers by rinsing down the drain.

2. Dispose of used conical tubes and pipettes into the biological waste bin.

3. Thoroughly rinse out the used bottle and hang up to dry.