免疫染色 (Immunostaining)

The English version follows the Japanese text.

準備

細胞（ガラス底ディッシュなどに播種）X samples

固定液

1 x PBSに1/10量のホルマリン（37%ホルムアルデヒド）を加えて、1xホルマリン溶液を作る。

2 x X sample (mL)

PBS

PBS + 0.2% Triton X-100 (peameabilization)

2 x X samples (mL)

PBS + 0.02% Triton X-100 + 3% BSA (blocking, 抗体希釈液)

2 x X samples + 0.4 x X samples (mL)

PBS + 0.05% Tween 20 (wash用、ただのPBSよりも乾きにくい）

1.5 x x X samples

1.　 mediumを捨てて細胞をPBSで1回wash

PBSなどのwash作業は、はがれやすい細胞などは気を付けてwashする。

複数のサンプルを同時に染色する場合には、1枚ずつ吸引、washをしたほうがいい。吸引して時間がたつと、サンプル（細胞）が乾燥してすぐにダメになる

2. 10% ホルマリン(final 3.7% holmaldehyde) in PBSで室温で15min静置、もしくは―30℃で氷冷したメタノールを 2 mL 加え、-30度の冷凍庫で15 min静置

抗体や染める対象によってどちらが良いか異なる。ホルマリンはタンパク質の華僑、メタノールは脱水固定。チューブリンはメタノールじゃないときれいに染まりにくい。

希釈率は抗体による。1/10 - 1/1000くらい。デフォルトは1/100くらい。

Can Get Signal Immunostainで希釈する場合もある。抗体によってはこれで劇的に変わる場合がある。

10. PBSに置換し、Imaging

Preparation

Cells seeded on glass-bottom dishes etc.

10% formalin in PBS (Fix solution)

Add 1/10 volume of formalin (37% formaldehyde) to PBS

2 mL for one 35 mm dish

PBS

0.2% Triton X-100 in PBS (Permeabilization solution)

0.02% Triton X-100 + 3% BSA in PBS (Blocking solution)

0.05% Tween 20 in PBS (Washing solution)

Procedure

1.　Remove medium and wash cells once with PBS

Note: Handle washing carefully for easily detached cells

When staining multiple samples, aspirate and wash one dish at a time to prevent sample drying

2. Choose one of the following fixation methods:

Option A: Incubate with 10% formalin (final 3.7% formaldehyde) in PBS for 15 min at room temperature

Option B: Add 2 mL ice-cold methanol (-30°C) and incubate for 15 min at -30°C

Note: Choice depends on antibody and target protein. Formalin crosslinks proteins, while methanol dehydrates. Tubulin typically stains better with methanol fixation.

3. Wash once with 1.5 mL PBS + 0.05% Tween 20, then add PBS + 0.2% Triton-X 100 and incubate for 15 min at room temperature (permeabilization)

4. Wash three times with 1.5 mL PBS + 0.05% Tween 20, then add PBS + 0.02% Triton X-100 + 3% BSA and shake for 1 hr at room temperature (blocking)

5. Dilute primary antibody in PBS + 0.02% Triton X-100 + 3% BSA and add 200 μL per 35 mm glass-bottom dish well. Incubate overnight at 4°C

Dilution ratio varies by antibody (typically 1/10 - 1/1000, default 1/100

Can Get Signal Immunostain solution may be used for dramatically improved results with some antibodies

6. Wash three times with 1.5 mL PBS + 0.05% Tween 20

7. Dilute secondary antibody (e.g., anti-Rabbit Alexa Fluor Plus-488) 1:1000 in 200 μL PBS + 0.02% Triton X-100 + 3% BSA and add to well. Shake for 1 hr protected from light

8. Wash three times with 1.5 mL PBS + 0.05% Tween 20

9. Optional: Add Alexa640-phalloidin or Hoechst 33258 (0.5 μL) or PI in 2 mL PBS and shake for ~5 min

10. Replace with PBS and proceed to imaging

Using 15mm coverslip and non-coated slide

Preparation

- Chemicals and reagents are same as above.

- Materials: Alcohol burner, tweezers.

- Preparing coverslip:

Put 1 pack of 15mm coverslip to 100ml B cup. Wash 3 times by water and gently shake (discard the powder on coverslip's surface). Remove water.

Wash by Ethanol 70% 3 times and gently shake. After washing, keep a proper Ethanol 70% in the cup with coverslip.

Prepare 35mm plastic dish, acohol burner, tweezers, 1 paper tissue (any types which does not leave any small particle after wiping is fine).

Spray Ethanol 70% to the tissue and put it on the surface of safety cabinet.

Using the tweezers to take 1 coverslip from the B cup with EtOH --> Dry on the paper (optional by fire ~ 2-3 seconds).

( Be careful not to fire it too long, the glass can be cracked )

Put the dry coverslip into 35mm dish (2-3 coverslip/35mm dish) then add the medium.

(Be careful not to overlap the coverslips and do not let the bubbles remain under any coverslip)

Seed the cell as usual.

Using EtOH and fire to sterile the tweezers and keep it in the safety cabinet.

Using the foil clean by EtOH to wrap the coverslip cup and maintain in 4°C

Procedure

Cut a Parafilm and firm it on the desk or a box with lid by RO water or EtOH 70% (Box if you incubate overnight)

Remove the medium and wash the cells with PBS

Fixation: Incubate the cells with 2mL 3.7% formaldehyde for 15 min at room temperature

Wash once with 1.5 mL of 0.05% Tween 20

Permeabilization: Incubate with 1.5 mL of 0.2% Triton-X 100 for 10-15 mins at room temperature

Wash three times with 1.5 mL of 0.05% Tween 20

Blocking: 100uL 0.02% Triton X-100 + 3% BSA. Incubate 10mins

From here: Using tweezers to separate the coverslips from the 35mm dish.

(Try not to touch the cell surface of the coverslips as much as possible. If you feel it is challenging, do not hesitate to ask me - Ngoc. "Not a conventional method but I know some tips to do that after several times harm the cells" :D)

Dilute the 1st antibody in 100uL 0.02% Triton X-100 + 3% BSA

50ul/coverslip: Drop 100ul 1st antibody/BSA to the parafilm and use the tweezers reversely put the cell-surface of the coverslip to the droplet. Close the lid (AVOID LIGHT) and incubate 30mins

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Wash the coverslips 3 times with 1.5 mL PBS + 0.05% Tween 20

Cut and firm parafilm --> Drop 3 droplets of PBS/Tween20 separately and put the cell-surface of coverslip on the top of droplet. Wait 2mins for each droplet then move to next droplet.

Dilute the 2nd antibody in 50uL 0.02% Triton X-100 + 3% BSA

50ul/coverslip: Drop 50ul 2nd antibody/BSA to the parafilm and use the tweezers reversely put the cell-surface of the coverslip to the droplet. Incubate 30mins (AVOID LIGHT)

Wash the coverslips 3 times with 1.5 mL PBS + 0.05% Tween 20 (same method as above)

Prepare non-coated slide (2-3 coverslips/slide)

Drop 15ul Mounting solution separately on slide

Wash coverslip with water: Prepare beaker with RO water and dip the coveslip properly into the water. Dry by touching a paper vertically with an angle of ~80°

Put the cell-surface of coverslip onto the Mounting solution droplet. Solidify in at least 20mins to 1hr in RT (AVOID LIGHT)

Proceed to imaging (If the surface of coverslip is cloudy, it may caused by dry salt. Clean by water is fine)

The immunostaining samples after imaging can be kept for several months in DARK condition (optional but better in 4°C)