SIV Brain Cutting Protocol

Always use universal precautions!

Wear lab coat or gown, gloves, face shield.

Equipment needed: surgical knife, safety scalpel, forceps, 4% paraformaldehyde in a jar to house brain, 2 ice buckets with dry ice, marker, pencil, freezer tubes, small tissue cassettes, 2 freezer boxes, large glass slides, O.C.T., frozen tissue cassette, and bleach.

Obtain CW# from database and pre-label freezer tubes, tissue cassettes, & freezer boxes.

IN HOOD: Hemisect the two hemispheres with a mid-sagittal cut through the corpus collosum and place the left side in 4% paraformaldehyde.

Make coronal cuts through the right hemisphere- approx. 1cm thick/ slice.

Chunks placed in freezer tubes should be approx. 0.1g. Freezer tubes should then be placed directly on dry ice.

Fill out frozen tissue freezer sheet. Relevant information should be entered in database after process is complete.

Right Hemisphere Processing

Make 3 O.C.T. sections for frozen sections- containing the following regions:

1. Cortical Gray and White Matter- CGM/CWM

2. Basal Ganglia- BG

3. Cerebellum- CB

Labeled freezer tubes: CW# +

5 with HI (Hippocampus)

5 with GP (Globus Pallidus)

5 with PUT (Putamen)

5 with CAU (Caudate)

10 with FC (Frontal Cortex)

10 with OC (Occipital Cortex)

10 with CB (Cerebellar Cortex)

10 with BS (Brainstem)

Left Hemisphere Processing

After 7 days of fixation, coronally section the left hemisphere.

Tissue Cassettes: labeled A-F (make G only if necessary)

A- Mid Frontal Cortex H- Spleen

B- Section with caudate I- Liver

C- Basal Ganglia J- Spinal Cord-cervical

D- Hippocampus, Thalamus K- Spinal Cord-thoracic

E- Cerebellum L- Thymus

F- Occipital Cortex M- Lymph node at sacrifice

G- Anything grossly weird N- Bone Marrow

O- Lung

P- Small bowel

Clean entire area and all equipment with bleach.

Block Identification for Monkey tissue processing

A- Mid Frontal Cortex

B- Section with Caudate

C- Basal Ganglia

D- Hippocampus, Thalamus

E- Cerebellum

F- Occipital Cortex

G- Anything grossly weird

H- Spleen

I- Liver

J- Spinal Cord-cervical

K- Spinal Cord-thoracic

L- Thymus

M- Lymph node at sacrifice

N- Bone Marrow

O- Lung

P- Small bowel

LN1 – 1st LN Bx of study

LN2 – 2nd LN Bx of study

LN3 – 3rd LN Bx of study

LN4 – 4th LN Bx of study

• Since each study has different times post-infection when LN Bx are performed, we have changed the block numbers below to LN1, LN2, etc. as of 6/2006. Q-T were used from 2002-2005 for sequential LN Bx and may not be the exact dates indicated below.

Q- Lymph node week 2

R- Lymph node week 4

S- Lymph node week 8

T- Lymph node week 12