Cryosectioning for Heart Fiber and Sheet Angles Calculation of a Rat Heart Protocol

Materials:

Single blade (razor blade)

Paper (post-its)

Super glue

Medium size cryomold

2-Methylbutane

Small/ Medium beaker

Dry Ice

Tweezers

Small scissors (optional to snip off excess tissue)

Needle

Small syringe

O.C.T

Sucrose and PSB (optional if use sucrose process)

Experimental Protocol:

*To make cryosectioning a little easier, apply the sucrose process before proceeding.

Cutting Transmural Slices

1. Cut transmural slices with a single slider. Cut a ring from the heart near the base perpendicular to the longitudinal axis at the left ventricle (LV). The blade is also perpendicular to the epicardium surface.

a. The circumferential direction is counter-clockwise if and only if the base faces out of the page and apex into the face.

b. Optional: Drive a needle along the longitudinal axis of the heart from the base to apex for visualization. The needle goes through the LV cavity between the non coronary cusp and the mitral valve.

2. Glue a thick paper (a couple of post-it notes thick) to the apex side, and leave a tail pointing to the right to show the +circ.

a. The thickness of a rat heart is about 1-3mm thick (use a ruler to measure)

3. Place the ring LV epi face down in a medium side cryomold against a side to line the ring from leaning (this bigger size cryomold makes the cryosectioning easier). Fill the cryomold with O.C.T.; if the sucrose process is being used, then fill it with the mixed 20%Sucrose and O.C.T solution.

4. Freeze the tissue by placing it in a beaker filled with 2-Methylbutane surrounded by dry ice (about 10 seconds) until it freezes.

5. Cyrosection this 1-2 section from the side of the block that embedded to the bottom of the cryomold.

6. To Cyrosection, squeeze a layer of O.C.T. to the metal mount and place the embedded tissue on the mount so the side to the bottom of the cryomold is facing away from the mount to you.

a. When slicing with the cryostat, trim the embedded sample until the shadow of the tissue can be seen.

b. When switching the amount of microns, the cryostat will take one more slice of the previous depth before proceeding to the amount changed.

7. Cut at about 10 microns and skip slices so that you get a total of 11 sections 10 microns thick each from the epicardium to the endocaridum. If this section of the heart is a little more than 2mm thick, then cut the tissue at 10 microns, skip 19 sections and section one more to keep. You continue sectioning like so until you get 11 sections.

8. Calculate the fiber angles between the papillary muscles.

Cutting Through-wall slices

1. At the LV between the papillary muscles, cut the bottom half of the heart into two using a single blade slicer (razor).

2. Use one of the pieces to cut 1-3 and the other to cut the 2-3.

3. To cut a 1-3 section, lay down the section and glue a piece of paper to the 2-3 side leaving a tail to the right (away from the right ventricle (RV)) to indicate the +circ direction.

a. Lay down the tissue on the 1-3 side that you want cyrosection into a cryomold

and repeat steps 4-6. Place this piece in the center of the cryomold. You only need one section from this piece of tissue.

b. Take measurements on the side where the LV is.

4. To cut a 2-3 section, lay 1-3 side down and glue it to a piece of paper. Leave a tail facing towards the right (towards the RV) to indicate the +circ direction.

a. Lay down the tissue on the 2-3 side that you want to cyrosection into a cryomold and repeat steps 4-6. Place this piece in the center of the cryomold. You only need one section from this piece of tissue

b. Take measurements on the side where the LV is.

Optional:

1. Staining

2. Cover slip

a. For adhesive, Permount may be used, but Cytoseal is less viscous and easier to use.

b. Squeeze a line of adhesive on the edge of the slide. Using a tweezer, slowly lower down the cover slip. Then, gently let go. This minimizes bubbles forming.

After cryosectioning, view sections under a light microscopy and capture digital pictures for calculating fiber and sheet angles on the computer.

Use the program “Image J” to calculate fiber angles (on 1-2 sections) and sheet angles. It can be downloaded for free here: http://rsbweb.nih.gov/ij/download.html
Sucrose Process: Preparing Fresh (or fixed) Heart Tissue for Cryosectioning

*** Picrosirius staining note ***

Washing in sucrose may cause increased background (red) staining of the muscle, obscuring the collagen fibers. It’s not a good idea to use this protocol while staining for collagen with PSR. (04/20/06, Darlene)

Solutions:

• 5% sucrose in 1x PBS
• 20% sucrose in 1x PBS
• Keep these in the refrigerator
• Discard if they start growing mold
• OCT

Before surgery:

• In a 15 ml conical tube, shake 3.5 ml 20% sucrose with 3.5 ml OCT
• This is enough to freeze one heart in one of the small circular cups.
• Pipet 10 ml 5% sucrose solution into a 15 ml tube
• Pipet 5 ml 5% sucrose and 5 ml 20% sucrose into a 15 ml tube
• Place all tubes in ice (if tissue is fresh)

Washes after surgery:

• 1st wash: 5% sucrose 10 min. (or until tissue sinks) on ice *
• 2^nd wash: 1:1 5%:20% sucrose 15 min. on ice
• Pre-mixed sucrose/OCT** 15 min. on ice
• The first 2 washes (5% then the 5%:20%) should be performed on a shaker
• Make sure the tissue sinks in solution before transferring it to the next solution
• Make sure there are no bubbles in the sucrose/OCT solution before embedding

Embed tissue in the sucrose/OCT solution by placing it a beaker filled with

2-methylbutane on top of dry ice.

*Notice “on ice” needed if only the tissue is fresh and not fixed

**The OCT/Sucrose is used for embedding

Notice: Since it may be hard to embed the tissue because of the sucrose in the solution, you can place the cryomold (or whatever container that you will be freezing the tissue in) directly on top of the dry ice and freeze layer by layer so that it is easier to orientate the tissue.

How to use the Cyrostat (Modified from http://www.bristol.ac.uk/vetpath/cpl/cryostat.html#Chuck):

• Switch on the internal light. The switch is down near the temperature gauge.
• Check that the temperature is at -20 degrees C, which is the standard operating temperature.  If you are cutting any specimen that has been formalin fixed is probably best cut at -10 degrees C. Harder tissues produce better sections at lower temperatures and vice versa (20 degrees is good enough though).
• Attach the tissue sample to the cork disc by putting a small amount of OCT onto the cork disk (metal mount) and placing the tissue sample on the OCT to freeze.
• Once the tissue is attached to the chuck it should be left in the cryostat for a little bit to freeze to the set temperature.

• Attach the chuck to the cryostat arm and tighten both wing nuts to hold it firmly.  Try to align the tissue so that it's flattest side is parallel to the knife edge.  This will help to achieve even cutting of the block and prevent sections from crumpling. Knobs can be found on the upper left side behind the cryostat arm.
• Adjust the section thickness using the micrometer screw on the left hand side of the cryostat.  Most tissues cut well at 6-8µm but you may have to increase this for difficult tissues. For our purposes, 10 µm is good enough.
• Trim the tissue block so that you have a complete section through the tissue.  This can be achieved by simply winding the main cutting handle (takes a long time) or by using the foot petal. Select a button for the path you want to choose for the cryostat arm to come to the blade. You can also adjust the speed.
• Remove all traces of tissue and ice from the knife-edge by brushing firmly UPWARDS with a stiff bristle paintbrush or using a knife swipe.
• The speed at which you turn the handle and cut the section depends on the type of tissue you are cutting.  Generally the harder the tissue the faster you need to cut it.  It is good to keep a couple consecutive slices so to get the best sections.
• Failure to cut sections is most often caused by a mis-aligned anti-roll plate. However, the anti-roll plate is often too hard to use. So, use two small and thin paintbrushes to guide the sections from the blade. Be careful to not touch the blade.
• To pick up the sections have a slide ready.  Use your fingers to place it quickly and evenly onto the tissue.
• Remove the slide and allow it to dry at room temperature for several minutes before you attempt any staining.
• Again brush off all remaining tissue and ice from the knife edge making sure you brush in an upward direction. If you use the knife wipe, then just slide it directly over the blade.
• Repeat as necessary.  When you are done remove all tissue waste from the machine and reset the advance mechanism to the top of its travel for the next user.
• Replace the insulating block and switch off the internal light.

To get the rat heart

• Anesthetize the rat by injecting them with 0.5 mL Ketamine
• Wait for it to be unresponsive
• Make an incision right underneath the sternum all the way across the belly
• Gently cut through the diaphragm
• Gently detach the connective tissue from ribs
• Cut up the ribs next to the sternum
• The aorta is underneath the fat pad and with the big tweezers, clamp the aorta and cut above your clamp
• Take out the heart and put it in saline to get all the blood out
• Cannulate aorta
• Perfuse with arrest solution 3-5 mL
• Perfuse with 10 mL of glut solution
• Store in glut solution