Differential staining of neuro tissue

Question 1

Preparation

Step 1a) & 1b)

Answer 1

 Immediately following post mortem the brain is removed and washed.
 It is then suspended by the Basilar Artery in 10% Formalin

Question 2

Preparation

Step 1c)

Answer 2

This process of suspension will take approximately 2 weeks.

Question 3

Preparation

Step 2a)

Answer 3

At the end of 2 weeks the brain is removed from the formalin and all the blood vessels on the surface of the brain are removed using a dissecting forcep.

Question 4

Preparation

Step 2b)

Answer 4

This is very important so that there is no interference of these vessels during slicing which will be done later.

Question 5

Preparation

Step 2c)

Answer 5

The brain is then washed again in cold water

and again immersed in 10% formalin for a further 4 weeks.

Question 6

Preparation

Step 4a)

Answer 6

After the 4 weeks the brain is removed and washed overnight in running water.

Question 7

Preparation

Step 4b)

Answer 7

25% aqueous solution of gelatin is prepared in warm water (30deg Celsius)and the brain is submerged briefly into this solution .

Question 8

Preparation

Step 4c)

Answer 8

The gelatin will prevent fragmentation of brain tissue during slicing.

Question 9

Preparation

Step 4d)

Answer 9

The brain is then left back into the formalin for 48 hrs.

Question 10

Slicing of the brain

Step 1:

Answer 10

Having decided on the plane in which the brain is to be sliced ( coronal ,sagittal or horizontal) , a cut is made midway using a sharp knife.

Question 11

Slicing of the brain

Step 2:

Answer 11

Bacon slicer is normally used and ideally the slices are 10-12mm thick.

Question 12

Slicing of the brain

Step 3 and step 4:

Answer 12

• Slices are then washed in running water and placed in 10% Formalin for 48hrs and this step is repeated.
• After washing the slices are ready for staining.

Question 13

Staining

Step 1:

Answer 13

During this stage the slices are handled very carefully with gloves to prevent any breakages.

Question 14

Staining

Step 2 and step 3:

Answer 14

 Staining can be done on a flat tray.
 Solution A , B and C are used for the staining
process.

Question 15

Solution A

Step 1:

Answer 15

Solution A comprises of dissolved 50gm phenol crystals and 5 gm copper sulphate in I L of distilled water containing 1.25ml HCl.

Question 16

Solution A

Step 2:

Answer 16

The brain slices are immersed in this Solution A for 5 min and optimal temperature of this solution is 60 deg Celsius.

Question 17

Solution A

Step 3:

Answer 17

After 5min in Solution A the slices are placed in ice water for 10 seconds.

Question 18

Solution B

Step 1:

Answer 18

After removing from ice water the slices are then placed in Solution B for 30-35 seconds.

Question 19

Solution B

Step 2:

Answer 19

Solution B comprises of Ferric chloride in distilled water.

Question 20

Solution B

Step 3:

Answer 20

After removal from Solution B the slices are then washed in running water for 1 minute

Question 21

Solution C

Step 1:

Answer 21

After removing from the running water the slices are then placed in Solution C. for 4 minutes.

Question 22

Solution C

Step 2:

Answer 22

Solution C comprises of 1% Potassium Ferrocyanide in distilled water.

Question 23

Solution C

Step 3:

Answer 23

After removal from Solution C the slices are placed overnight in running water.

Question 24

Results

Step 1:

Answer 24

The grey matter will appear blue with gelatin appearing darker blue on the outside.

Question 25

Results

Step 2 and step 3:

Answer 25

 The white matter will appear as very light blue

 The slices can be then be mounted for display.