Hydrogel Solution Incubation

From CLARITY Wiki

(Difference between revisions)
Jump to: navigation, search
Line 2: Line 2:
   
 
===Importance of Hydrogel Solution Incubation===
 
===Importance of Hydrogel Solution Incubation===
  +
Allowing ample time for the hydrogel monomers to distribute uniformly throughout the tissue sample is important for incorporating biomolecules into the hydrogel network during the tissue embedding step.
  +
 
(Paragraph about the role of formaldehyde and acrylamide in the tissue - crosslinking with primary amine groups on biomolecules; explain that changes to the hydrogel solution may alter what gets crosslinked into the hydrogel, possibly resulting in more protein loss; show "step 1" picture from paper; explain that fixed tissue has already undergone crosslinking by formaldehyde, but results on fixed human samples show that enough unused sights remain for clarity, but increased incubation times are used to allow more time for monomers to find these sites)
 
(Paragraph about the role of formaldehyde and acrylamide in the tissue - crosslinking with primary amine groups on biomolecules; explain that changes to the hydrogel solution may alter what gets crosslinked into the hydrogel, possibly resulting in more protein loss; show "step 1" picture from paper; explain that fixed tissue has already undergone crosslinking by formaldehyde, but results on fixed human samples show that enough unused sights remain for clarity, but increased incubation times are used to allow more time for monomers to find these sites)
   

Revision as of 00:35, 7 April 2014

Tissue samples are placed into the hydrogel solution to incubate for several days. This allows the hydrogel monomers, formaldehyde and acrylamide, to diffuse uniformly throughout the tissue. Perfusion is recommended because it introduces the monomers inside the tissue prior to incubation.

Contents

Importance of Hydrogel Solution Incubation

Allowing ample time for the hydrogel monomers to distribute uniformly throughout the tissue sample is important for incorporating biomolecules into the hydrogel network during the tissue embedding step.

(Paragraph about the role of formaldehyde and acrylamide in the tissue - crosslinking with primary amine groups on biomolecules; explain that changes to the hydrogel solution may alter what gets crosslinked into the hydrogel, possibly resulting in more protein loss; show "step 1" picture from paper; explain that fixed tissue has already undergone crosslinking by formaldehyde, but results on fixed human samples show that enough unused sights remain for clarity, but increased incubation times are used to allow more time for monomers to find these sites)

CLARITY can still be performed on tissue samples that have not been perfused with hydrogel solution.

Unfixed or Fixed Tissue Incubation

Whenever possible, it is best to replace the normal fixative solution with the hydrogel monomer solution, even when perfusion is not performed. However, tissue that has been stored in fixative solution, such as formalin, can also be processed using CLARITY. Place non-perfused tissue in the ice cold hydrogel solution (freshly-made or thawed) and incubate at 4°C in the refrigerator. Incubation can be on a shaker plate or standing still. The amount of hydrogel solution used should be enough to completely cover the tissue sample and provide a small excess to support monomer diffusion into the tissue.

Incubation Time

The time period of incubation in the hydrogel monomer solution for non-perfused tissue samples depends on the size of the sample and whether it was previously fixed. For unfixed tissue, smaller samples (e.g., less than 1-2 mm thick) may only need 2-3 days of incubation before embedding, while larger samples, like a whole mouse brain, may require longer soaking time, such as 5-7 days or more. Fixed tissue samples have already undergone crosslinking of many of the primary amine sites within the cells, causing less sites to be readily available for crosslinking with the formaldehyde and acrylamide hydrogel monomers. Longer incubation times, such as 1-2 weeks, should be used for samples previously stored in fixative to allow extra penetration time for the monomers to diffuse into the crosslinked tissue and find the unused primary amine sites for hydrogel attachment.

Sample Storage

Tissue samples should not be stored in the hydrogel solution at 4°C for more than 2-3 weeks. The thermal initiator in the hydrogel solution is not stable for long periods of time at this temperature, and acrylamide polymerization can slowly occur. It is undesirable to let polymerization occur uncontrolled over time as it could lead to an excessive formation of crosslinked polyacrylamide inside the tissue, rendering it rigid and difficult to clear (see Hydrogel Embedding).