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Schuele Lab Instructional Protocols

Immunostaining for Three Germ Layers with Antibodies against β-III-tubulin, AFP, and SMA  

Purpose:

The purpose of the protocol is to confirm that regenerated iPSC colonies can spontaneously differentiate into three germ layers: ectoderm, endoderm, and mesoderm. By immunostaining the differentiated cells with antibodies against β-III-tubulin, Smooth muscle actin (SMA), and Alpha feto protein (AFP), we can verify their ability to develop into all three germ layers.

 

Materials:

  • Slides à24-well plates/ 8 well chamber slides (Fisher Scientific, # 1256518)
  • Geltrex (Life Technologies, A1413302)
  • P20/P200/P1000 pipetman
  • Lab rotator/shaker (Fisher Scientific, # 2314)
  • 1X Phosphate buffer saline (PBS) with Ca++ and Mg++ (Fisher Scientific,SH30264.02)
  • 15ml Falcon tubes
  • 20% paraformaldyhide (PFA) (Electron microscopy sciences, 15713)
  • Triton X-100 (Sigma, # S100)
  • 100% Glycerol (BioRad, # 9455)
  • Three primary Abs
  • Secondary Abs
  • 100% normal goat serum (Fisher Scientific,# 101098-382) or serum from host of secondary antibody (Ab)
  • 10mg/ml Hoechst (Life Technologies, # H3570) or 10mg/ml DAPI (Life Technologies, # D3571)
  • Cover slips (Fisher Scientific, # 174950)
  • 100% normal goat serum (Fisher Scientific,# 101098-382) or serum from host of secondary antibody (Ab)
  • Clear nail polish

 

Preparation of working solutions:

  1. Primary antibody

    Antibody

    Germ Layer

    Host

    Isotype

    Manufacturer

    Catalog #

    Dilution

    AFP

    Endoderm

    Mouse Monoclonal

    IgG2b

    Sigma

    SAB3300008

     

    1:300

    SMA

    Mesoderm

    Rabbit Monoclonal

    IgG

    Millipore

    MAB041094

     

    1:500

    β-III-tubulin

    Ectoderm

    Mouse Monoclonal

    IgG2a

    Covance

    MMS-435P

    1:300

  2. Secondary antibody

Antibody

Host

Isotype

Manufacturer

Catalog #

Dilution

 

 

2. Secondary antibody:

Antibody

Host

Isotype

Manufacturer

Catalog #

Dilution

Allophycocyanin®

Goat

IgG

Life Technologies

A865

1:200

Alexa Fluor® 488

Goat

IgG

Life Technologies

A11029

1:200

 

 

 

 

3. Blocking Buffer: 5% normal goat serum [fz1]  diluted in PBS.

To make 10ml blocking buffer, combine 500ul normal Goat serum + 9.5ml PBS.

 

4. 4% PFA diluted down from 20% stock PFA to fix the cells.

To make 10ml of 4%PFA, add 2ml of 20%PFA to 8ml PBS.

 

5. Permeabilize solution: 0.3% Triton X-100 in 5% goat serum.

 

6. 1µg/ml of Hoechst diluted in PBS from 10mg/ml stock to counter stain nucleus.

 

Method:

 

A.                Growing Embryoid Bodies (EB) in Slide Before Immunostaining:

 

1.                  Coat slides/plates with 1:100 geltrex.

2.                  Culture two/three EBs made from one of the iPSC colonies in each well of an 8-well chamber slides with media for ten days.

3.                  On the day of staining, aspirate off the media and rinse wells once with 300µl of 1X PBS.

4.  Keep the cells hydrated with 1X PBS before fixation.

 

B.                 Fixation:

 

5.                  Aspirate off PBS from the wells.

6.                   Fix the cells with 100µl of 4% PFA per well at room temperature (RT) for ten minutes.  

7.                   Aspirate off the 4% PFA [fz2] and wash[fz3]  three times with 300µl of 1X PBS for five minutes each time on the shaker.

8.                   Permeabilize[fz4]  cells with 200µl of 0.3% Triton X-100 in 5% goat serum per well for five minutes.

9.                   Wash wells once with 1X PBS for five minutes.  

10.   Add 200µl of blocking buffer to all wells and incubate at RT for 60 minutes on the shaker. 

11.  Aspirate off the blocking buffer.

 

C.                Ab Staining:

 

12.   Add 300µl of 1º monoclonal Ab (mAb) to the corresponding wells (except in the control well for 1º Ab):

  To make 500µl of SMA à 1.00µl SMA + 499.0µl blocking buffer (1:500)

  To make 500µl of AFP à 1.0µl AFP+ 499.0µl blocking buffer (1:300)

  To make 500µl of β-III-tubulin à 1.67µl Oct4 + 498.33µl blocking buffer (1:300)

 

 

 

 

 

 

 

 

 

 

 

Example layout of three germ layers’ markers in 8-well chamber slide for each clone:

 

β-III-tubulin

1˚ Ab: human α- β-III-tubulin mouse Ab (1:300)

2˚ Ab: Allophycocyanim goat α-mouse IgG (1:200)

Hoechst

Expect: Red

 

Control for 1˚ Ab

1˚ Ab: no mAb, only PBS

2˚ Ab: Allophycocyanim goat α-mouse IgG (1:200)

Hoechst

Expect: No Red

 

AFP

1˚ Ab: human α-AFP mouse Ab (1:300)

2˚ Ab: Allophycocyanim goat α-mouse IgG

Hoechst

Expect: Red

 

No cells

Empty

SMA

1˚ Ab: human α-SMA mouse Ab (1:500)

2˚ Ab: Allophycocyanim goat α-mouse IgG (1:200)

Hoechst

Expect: Red

 

Control for 2˚ Ab

1˚ Ab: human α-SMA mouse Ab (1:500)

2˚ Ab: no Ab, only PBS

Hoechst

Expect: No Red

 

No cells

Empty

No cells

                     Empty

 

 

13.   Incubate at RT for 1-2 hours or at 4ºC overnight[fz5] .

14.   Wash wells three times with 1X PBS on the rotator at lowest speed for five minutes each time.

15.   Prepare1:200 goat-anti-mouse secondary Ab:

To make 1000µl of goat-anti-mouse secondary Ab à 5.0µl Ab + 995µl blocking    buffer.

16.   Add 400µl of secondary antibodies, Allophycocyanin goat α-mouse IgG to all wells except in control well for secondary Ab.

17.   Incubate in the dark (cover with aluminum foil) at RT for one hour on the shaker.

18.   Wash wells once with 1X PBS and covered chambered slides with aluminum foil.

19.   Prepare 1ug/mL Hoechst and incubate each well with 150µl of Hoechst in the dark for 3 minutes.

20.  Wash wells six times [fz6] with 1X PBS.

21.   Aspirate of all the PBS and take the chambers out [fz7] from the slides.

22.   Put ~300µl mounting solution (80% glycerol + 20%PBS) and mount the slides with cover slips. Then seal the outside with nail polish.

23.  Use fluorescence microscopy to view the images.


 [fz1]Blocking buffer should be the same species as the secondary Ab is raised.

 [fz2]Used up PFA should be discarded in a labeled biohazard container. (See figure2 Misc\PFA waste container_1.jpg)

 [fz3]Do Not dry off wells while washing. Always keep enough PBS to cover surface of the wells. (See figure3 Misc\8-well chamber slide with minimum amount (100ul) of PBS.jpg)

 

 [fz4]Colonies that will be stained with nuclear markers need to be permeabilized. DO NOT permeabilize cells if using a surface marker. You do not want to poke a hole on the cell membrane, if you are staining for surface markerJ.

 [fz5]Based on my experience, overnight incubation at 4ºC deliver better results.

 [fz6]Slides are ready to view at this point. Important note à always keep the wells hydrated with PBS before mounting, otherwise the cells are going to dry off.

 [fz7]Follow directions on chambered well slide’s packet and be gentle not to disturb cells.

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