PURPOSE: To stain the slides with haematoxylin and eosin
Materials:
- Slides with tissue sections
- Haematoxylin solution Gill Modified solution3 (Order at VWR Harleco Cat N 65067)
- Eosin Y, solution 1% alcoholic solution (Order at VWR Harleco Cat N 588X)
- 100% EtOH
- 95% EtOH
- 80% EtOH
- Xylene
- Slides racks
- Dry dishes (there must not be water in the different dishes you use for the xylene and 100%EtOH)
- Permanent mounting media
- Coverslips
- Forceps
- Gauze sponge
- Q-tips
Procedure: the procedure involves moving the slides from solution to solution in different staining dishes
Step 1: Deparaffinizing the slides
a) Xylene for 5 minutes
b) Xylene for 1 minute
c) 100% EtOH for 1 minute
d) 100% EtOH for 30 seconds
e) 95% EtOH for 1 minute
f) 95% EtOH for 30 seconds
g) 80% EtOH for 1 minute
h) Tap water rinse for 1 minute
Step 2: Staining with haematoxylin
a) Haematoxylin 1 dip!
b) Go quickly to tap water rinse for 2 minutes
c) 1% acid solution (2mL of Acid Acetic + 98 mL of Disyilled water) : 10dips
d) Running tap water for 2 to 5 minutes
Step 3: Counterstain
1) Eosin for 2-3 minutes
Step 4: Dehydrating
a) 95% EtOH for 1 minute
b) 100% EtOH for 1 minute (use a new EtOH solution)
c) 100% EtOH for 30 seconds
d) Xylene for 1 minute (use a new xylene solution)
e) Xylene for 30 seconds
f) Leave slides in Xylene until coverslipping
Now the slides are ready to be coverslipped to preserve them.
Step 5 : To coverslip the samples to preserve them
a) Remove the slides from the xylene
b) With a gauze sponge wipe the excess clearing agent from the slide to approximately 2-3 mm from the margin of the specimen. If specimen are close to the edge of the slide, Q-tips are very useful to map up xylene
c) Using the transfer pipette, place approximately two-four drops of mounting media in the center of the slide. There must be enough medium to fill the space between the cover glass and the slide.
d) Place the edge of the cover glass on the edge of the slide over the center of the specimen
e) Gradually bring the cover glass and slide together, drawing the medium evenly over the specimen.
f) Remove wet mounting media excess with Q-tips.
g) Remove excess of mounting media with xylene.
If air bubbles occur under the cover glass tease them out by gentle tapping the cover glass with a dissection needle or forceps. If the bubbles cannot be removed soak the slide for a minute or two in clearing agent (xylene) and repeat steps 1-7.