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24578v2-long-narrowsmall.gif (101597 bytes)Sample Preparation for Electron Microscopy

The AIF staff will process your samples after you perform the primary fixation with fixative that we supply.  The AIF staff has reserved Wednesday and Thursday for embedding for TEM and critical point drying for SEM.  In order to accommodate a large number of samples from different laboratories, the following guidelines must be followed:

FIRST, speak with a member of the facility to plan your experiment.  Speak to Frank Macaluso or Leslie Gunther-Cummins.  You can e-mail or phone any of the above people.  Please meet with one of us before you bring samples for processing!

SECOND, ALL SAMPLES FOR TEM OR SEM MUST BE IN THE AIF BY WEDNESDAY AT 11:00 AM!

The following protocols work well for most mammalian tissue and mammalian cells in tissue culture.  Certain cell types require different fixation protocols, so please meet with the AIF staff to be sure that you are going to use the proper fixation protocol for your experiment.

****  If you wish to look at pathogenic or infectious agents, you must speak to a staff member in advance.  Fixation protocols will be extended and/or otherwise customized.  ****

TEM fixation of tissue for morphology TEM fixation of cells in monolayer culture for morphology
TEM fixation of tissue for immunolabelling TEM fixation of cells in monolayer culture for immunolabelling
SEM fixation of Tissue SEM fixation of cells in culture

TEM fixation of tissue for morphology:

1.      Sacrifice your animal.  Sacrifice one animal at a time!

2.      Quickly excise the tissue of interest.

3.      Place immediately into a Petri dish containing 2% paraformaldehyde + 2.5% gluteraldehyde in 0.1M Cacodylate buffer.  The AIF staff will provide this for you.

4.      Cut tissue into small cubes. The cubes must have one dimension that is 1mm, the other dimensions may be slightly larger.

5.      Place 5 or 6 tissue cubes into a labeled vial containing fresh 2% paraformaldehyde + 2.5% gluteraldehyde in 0.1M Cacodylate buffer.

6.      Fix tissue for 60 minutes at room temperature. 

7.      If it is early enough, bring tissue to AIF, room 639 in fixative.   Be sure to speak to someone before leaving tissue!

8.      If it is the end of the day, replace fixative with 0.1M Cacodylate buffer and store overnight at 4 (in the fridge).

9.      Bring tissue to AIF, room F-639, in buffer.  Be sure to speak to someone before leaving tissue!

ALL TISSUE SAMPLES MUST BE BROUGHT TO THE AIF AS CUT UP CUBES.

ALL TISSUE SAMPLES MUST BE BROUGHT TO THE AIF BY 11:00 AM ON WEDNESDAY.

Any samples not making this cut off deadline will be held for processing until the following Wednesday.


TEM fixation of cells in monolayer culture for morphology:

1.      Grow your cells in plastic Petri dishes.  We will accept any size dish, but prefer 60mm.  We also prefer duplicate dishes.

2.      Do fixation protocol one dish at a time!  Do not let cells dry out!

3.      Rinse cells for 1 minute with serum free media at the incubation temperature.

4.      Pipette off the serum free media.

5.      Immediately add 2% paraformaldehyde + 2.5% gluteraldehyde in 0.1M Cacodylate buffer at room temperature.  Gently add this from the edge of the dish.  The AIF staff will provide this solution for you.

6.      Fix dish for 20 to 30 minutes at room temperature.

7.      Bring sample to AIF, room F-639, in fixative.  Be sure to speak to someone before leaving cells!

ALL CELLS IN CULTURE MUST BE BROUGHT TO THE AIF BY WEDNESDAY AT 11 AM.  If cells require an additional day of growth for optimal sampling, we will accept monolayers on Thursday by 11:00 AM.  This is the only exception to the Wednesday 11:00 AM rule and must be arraigned in advance.

Any cells not making the Thursday cut off deadline will be held for processing until the following Wednesday.


TEM fixation of tissue for immunolabelling:

1.      Sacrifice your animal.  Sacrifice one animal at a time!

2.      Quickly excise the tissue of interest.

3.      Place immediately into a Petri dish containing 4% Paraformaldehyde + 0.1%gluteraldehyde in 0.1M Cacodylate buffer.  The AIF staff will provide this for you.

4.      Cut tissue into small cubes. The cubes must have one dimension that is 1mm, the other dimensions may be slightly larger.

5.      Place 5 or 6 tissue cubes into a labeled vial containing fresh 4% Paraformaldehyde, 0.1%gluteraldehyde in 0.1M Cacodylate buffer.

6.      Fix tissue for 60 minutes at room temperature.

7.      If it is early enough, bring tissue to AIF, room F-639, in fixative.   Be sure to speak to someone before leaving tissue!

8.      If it is the end of the day, replace fixative with 0.1M Cacodylate buffer and store overnight at 4 (in the fridge).

9.      Bring tissue to AIF, room F-639, in buffer.  Be sure to speak to someone before leaving tissue!

ALL TISSUE SAMPLES MUST BE BROUGHT TO THE AIF AS CUT UP CUBES.

ALL TISSUE SAMPLES MUST BE BROUGHT TO THE AIF BY 11:00 AM ON WEDNESDAY. 

Any samples not making this cut off deadline will be held for processing until the following Wednesday.


TEM fixation of cells in monolayer culture for immunolabelling:

1.      Grow your cells in plastic Petri dishes.  We will accept any size dish, but prefer 60mm.  We also prefer duplicate dishes.

2.      Do fixation protocol one dish at a time!  Do not let cells dry out!

3.      Rinse cells for 1 minute with serum free media at the incubation temperature.

4.      Pipette off the serum free media.

5.      Immediately add 4% Paraformaldehyde, 0.1%gluteraldehyde in 0.1M Cacodylate buffer at room temperature.  Gently add this from the edge of the dish.  The AIF staff will provide this solution for you.

6.      Fix dish for 20 to 30 minutes at room temperature.

7.      Bring sample to AIF, room F-639, in fixative.  Be sure to speak to someone before leaving cells!

ALL CELLS IN CULTURE MUST BE BROUGHT TO THE AIF BY WEDNESDAY AT 11 AM.  If cells require an additional day of growth for optimal sampling, we will accept monolayers on Thursday by 11:00 AM.  This is the only exception to the Wednesday 11:00 AM rule and must be arranged in advance.

Any cells not making the Thursday cut off deadline, will be held for processing until the following Wednesday.


SEM fixation of Tissue:

1.      Sacrifice your animal.  Sacrifice one animal at a time!

2.      Quickly excise the tissue of interest.

3.      Place immediately into a Petri dish containing 2.5% gluteraldehyde in 0.1M Cacodylate buffer with MgCl + sucrose.  The AIF staff will provide this for you.

4.      Cut tissue into areas of interest.  Do not hold an area of interest with forceps!  This will show as mechanical damage to your tissue in the SEM.

5.      Place tissue of interest into a labeled vial containing fresh 2.5% gluteraldehyde in 0.1M Cacodylate buffer.

6.      Fix tissue for 60 minutes at room temperature. 

7.      If it is early enough, bring tissue to AIF, room 639 in fixative.   Be sure to speak to someone before leaving tissue!

8.      If it is the end of the day, replace fixative with 0.1M Cacodylate buffer and store overnight at 4 (in the fridge).

9.      Bring tissue to AIF, room F-639, in buffer.  Be sure to speak to someone before leaving tissue!

ALL SEM SAMPLES MUST BE BROUGHT TO THE AIF BY WEDNESDAY AT 11:00 AM.


SEM fixation of cells in culture:

1.      Grow your cells on glass 13 mm round coverslips in plastic Petri dishes.  The AIF staff will provide these for you.  You must use the coverslips provided by the AIF.   They must be cleaned and sterilized before plating out your cells.

2.      Do fixation protocol one dish at a time!  Do not let cells dry!

3.      Give your cells a serum free media rinse.  This washes out any proteins that may be in the dish.

4.      Pipette off the serum free media.

5.      Immediately add 2.5% gluteraldehyde in 0.1M Cacodylate buffer.  Gently add this from the edge of the dish.  The AIF staff will provide this solution for you.

6.      Fix dish for 20 to 30 minutes at room temperature.

7.      Bring sample to AIF, room F-639, in fixative.  Be sure to speak to someone before leaving cells!

 ALL SEM SAMPLES MUST BE BROUGHT TO THE AIF BY WEDNESDAY AT 11:00 AM


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