Woodmansee Lab Standard Procedure
Staining Coverslips with Iron Hematoxylin
Investigator: ______Date: ______Protocol: ______
q Locate or prepare each of the following solutions.
q 25% glacial acetic acid / 75% ethanol (5 ml/coverslip)
q 70% ethanol (the stuff in the squirt bottle is fine)
q Working Hematoxylin Solution (4-5 ml/coverslip)
q Tap water (I really mean TAP water, not the good stuff)
q 95% ethanol (we usually have a bottle of this in the lab)
q 100% ethanol (Use the bottle with the beads in the bottom).
q 100% Xylene (Stored in the flammable cabinet)
q Permount (stored in the flammable cabinet)
q Remove the coverslip from the cell culture dish and immediately place it in a small glass petri dish that contains 5 ml of the acetic acid/ethanol solution. Leave it in this solution for at least 5 minutes. You may also wrap up the dish tightly with Parafilm and keep it till you have time to run the rest of the procedure.
q Draw off the acetic acid/alcohol with a pipet and replace it with 4-5 ml of 70% ethanol. Keep this solution in the dish for 5 min.
q Replace the ethanol with fresh 70% ethanol, hold for another 5 minutes.
q Replace the ethanol one more time with fresh 70% ethanol, hold for another 5 minutes.
q Remove the alcohol and fill the dish with tap water. Hold for 5 minutes
q Replace the water with Working Hematoxylin for 4 min.
q CAREFULLY rinse the coverslip with running tap water till the water runs clear. Be careful not to wash the coverslip out of the dish or to flip the coverslip over in the swirling water. Hold in water for an additional 5 min.
q Replace water with 70% ethanol for 5 min.
q Replace 70% ethanol with 95% ethanol for 5 min.
q Replace 95% ethanol with 100% ethanol for 5 min.
q Replace ethanol with Xylene for 5 min (do this in a fume hood).
q Use a lead pencil to mark the frosted end of a clean microscope slide with complete identifying information.
q Place a drop of Permount on a clean microscope slide and place the stained coverslip on the Permount CELL SIDE DOWN.
q Use a small brush to paint Permount over the pencil marks so they can not get rubbed off.
q Discard Xylene in the “Used Organic Solvents” jug.
q Allow slides to dry at least overnight.
Notes:
· DO NOT ALLOW COVERSLIPS TO DRY OUT AT ANY POINT IN THIS PROCEDURE.
· Recipe for Working Iron Hematoxylin Stain:
1 part Stock Hematoxylin Stain
1 part Iron Alum Solution
Make new working stain weekly
· Recipe for stock Hematoxylin Stain:
5 g Hematoxylin powder
500 ml 100% ethanol
Allow stain to "ripen" for a least a week before use.
· Recipe for Iron Alum Solution:
5 g ferrous ammonium sulfate
5 g ferric ammonium sulfate
5 ml concentrated HCl
Distilled water to 500 ml
· Permount is Fisher Scientific Catalog # SP15-100.
· 100 % Ethanol should be stored in a bottle with a few inches of molecular sieves (Fisher #M564-500) to keep ethanol completely dehydrated. Pour carefully out of bottle to keep fine particles of beads from getting into the petri dish.
· This procedure was revised 20-Feb-04.