Version 0, July 4, 2016 Stock Solutions 4 Paraformaldehyde



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Version 1.0, July 4, 2016

Stock Solutions

4 % Paraformaldehyde

4 % Paraformaldehyde (from Electron Microscopy Science 16% stock)

1x PBS
Permeabilization Buffer (Store at 4 C, can be used over an extended period of time)

1x PBS


0.1% Triton X-100
Cell Blocking Buffer (Store at 4 C, can be used for at least 1 month)

1x PBS


5% normal donkey serum

Monomer solution:

Component

Stock concentration*

Amount (mL)

Final concentration*

Sodium acrylate**

38

2.25

8.6

Acrylamide

50

0.5

2.5

N,N′-Methylenebisacrylamide

2

0.75

0.15

Sodium chloride

29.2

4

11.7

PBS

10x

1

1x

Water




0.9




Total




9.4**




*All concentrations in g/100 mL except PBS

**9.4/10 mL (1.06x), the remaining 6% volume brought up by initiator, accelerator and inhibitor as needed (see below).



Materials and Stock Solution Storage:

Sodium acrylate sometimes comes with a variable purity level, which can affect performance. For every fresh bottle purchased, we make a 38g/100mL (33 wt%) sodium acrylate stock and check to insure that it is colorless under normal room lighting. If the stock has a yellow tint (see Figure 1, below), we discard the bottle from which it was made. Once open, we have been storing sodium acrylate in an airtight, low humidity, or dessicator chamber in -20 degrees C, since the solid is moisture sensitive. APS powder and 100% TEMED solution are stored in a room temperature dessicator.





Figure 1. 38g/100mL Sodium Acrylate stock solutions: correct (left) and low purity (right).

We have been storing the monomer solution mixed up at -20 degrees C for up to 1 month. TEMED, APS, and H-Tempo Stock solutions can be kept in -20 degrees C, and we generally remake the TEMED and APS stocks at least once every 2 weeks.



Gelation Solution: Mix the following 4 solutions on ice. Monomer solutions + TEMED accelerator + APS initiator solution + Water (Initiator solution needs to be added last to prevent premature gelation). Solutions should to be vortexed to ensure fully mixing.
For 200 µL gelling solution, mix the following:

Monomer solution (1.06x) (188µl) (keep at 4C to prevent premature gelation):

Accelerator solution (4µl): TEMED (TEMED stock solution at 10%, final concentration 0.2% (w/w). (Accelerates radical generation by APS).

Initiator solution (4µl): APS (APS stock at 10%, final concentration 0.2% (w/w)). (This initiates the gelling process. This needs to be added last).

Inhibitor solution: not needed for cells, bring up to 200 µL with ddH2O (4 µL).

Digestion Buffer (can be stored as aliquots in freezer at -20C):

50 mM Tris pH 8.0

1 mM EDTA,

0.5% Triton X-100,

0.8 M guanidine HCl (8M guanidine HCl stock solution can be kept at RT)

Add:


Proteinase K (1:100, final concentration 8 units/mL) to digestion buffer before use.
ExM procedures for cultured cells

Cell Culture:

Cells can be grown as desired to suit experimental needs. However, for ease of gelation and imaging, and handling, we have found 16 well Culturewells removable chambered coverglass from Grace Bio Labs (Sigma catalog: GBL112358-8EA) to be great for before imaging, as well as subsequent gelation and digestion.



Fixation:

Fixation and permeabilization conditions will depend on immunostaining protocol, we have tried the procedure with PFA as well as PFA/glutaraldehyde. Below is a standard fixation and permeabilization protocol.



  1. Fix with cold 4% PFA for 10 min at RT.

  2. Wash with 5 min 1x PBS + 100 mM glycine to quench fixation.

  3. Wash 2x 5 min with 1x PBS

  4. Permeabilize with permeabilization buffer at RT for 15 minutes.

Primary antibody staining: Essentially the same as conventional histology.

  1. Block cells with blocking buffer for 15 minutes

  2. Incubate cells with primary antibodies in blocking buffer at desired primary concentration.

    1. Can do incubation from 1 hr to overnight at RT/ 4 C.

  3. Wash 4x 5 min with PBS.

Secondary Antibody Staining

  1. Incubate slice with secondary antibodies in blocking buffer for 2-4 hours at RT or overnight at 4C on a shaker.

    1. Note: Do not use cyanine derivatives: (Cy3, Cy5, Alexa 647) on your secondary antibodies. We suggest using CF633 (from Biotium) for far red staining.

  2. Wash slices with blocking buffer, 4 times, ~5 min each.

Anchoring treatment:

  1. Re-suspend Acryloyl X – SE (Life technologies: A20770) in 500 uL anhydrous DMSO (10 mg/mL stock solution). Aliquot in 20 uL increments, and store desiccated at -20 C.

  2. Dilute AcX 1:100 (0.1 mg/mL) in 1x PBS.

  3. Treat stained cells or cells expressing genetically encoded FPs for > 6 hours at RT (this reaction can be left overnight).

  4. Wash 2x 15 minutes each before proceeding to gelation. Samples can be stored at this point at 4 C.

Gelation:

Expansion speed is generally limited by the diffusion time of salt and water out/into the gel, thus casting thin gels will generally speed up expansion time. The Grace Bio labs removable coverglass has a removable chamber upper structure which can be removed via a removal tool (Sigma catalog: GBL103259, see Figure 2 below). After removal of the chamber, a 1 mm silicone spacer remains with the coverslip and can be used to cast the gel (protocol described below)1. If cells are grown on coverglass, then a chamber similar to the slice gelation chamber can be made using coverslip spacers (see “ExM 1.0 Brain Slice Protocol”).



  1. Remove upper structure from the Grace Bio labs chamber leaving silicone gasket on coverslip.

    1. It helps to remove most of the liquid from each well before doing this; leave ~40 uL of PBS in each well.

  2. Prepare a glass slide covered with parafilm to use as the top of the gelation chambers.

    1. The parafilm prevents the gels from sticking to the top glass slide.

  3. Prepare gelation solution on ice (see above section), adding water and TEMED accelerator to monomer solution; do not add initiator yet.

    1. The volume of each well with 1 mm silicone gasket is ~ 40 uL; prepare 45 uL of gelation solution for each well.

  4. Remove remaining PBS from the wells, make sure to remove all PBS as remaining PBS will dilute gelation solution.

  5. Add APS to gelation solution, vortex briefly, and immediately pipette 40 uL of gelation solution into each silicone gasket well.

  6. Carefully place the parafilm glass slide (parafilm facing down) on top of silicone gasket to seal the gelation solution within the wells.

    1. Done correctly, there should be no bubbles within each well and the gelation solution will completely fill the volume.

  7. Protect sample from dark and move to 37 C incubator for 1 hour for gelation.



Figure 2. Grace bio labs culturewell, with removal tool showing upper structure and bottom silicone gaskets on coverglass after disassembly (reproduced from Sigma catalog page).

Digestion and Expansion:

  1. Digest cells with digesting buffer, overnight @ room temperature. (Make sure at least 10-fold excess volume of digestion buffer is used.) With Grace Biolabs chambers:

    1. After gelation, carefully remove parafilm slide from top.

    2. Then remove black silicone gasket, but keep gels remaining on glass. The black silicone gasket should peel off easily with tweezers or by hand.

      1. Gels will pop off glass surface during digestion on their own; don’t peel the gels off the surface of the glass.

  2. Wash cell gels with excess volume of ddH2O (we usually use at least 10x the final gel volume), 3-5 times, for 1 hour each time. Gel expansion reaches plateau after about the 3rd or 4th wash.

  3. Gels can be expanded in a flat plastic dish, such as a petri dish, or directly in the chamber that you wish to image in. The expansion chamber needs to be of adequate size for the sample, we found that for the culturewell gels, the expanded gel fits nicely in a glass bottom 6 well plate (In Vitro Scientific). Gels can be immobilized with 2% low melt agarose in water to prevent drift during imaging.

Then image with conventional fluorescent, confocal microscope, or other desired scopes.

1 We have found that the top structure can also be removed using a razor blade if one does not have a removal tool.


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