Immunohistochemistry (IHC) in mouse brain tissue

This protocol enables detection of proteins of interest using specific antibodies in perfusion-fixed mouse brain sections.
 

Preparation of mouse brain tissue:

1)  Inject mice of desired age (postnatal day 3 and on) with the appropriate volume of ketamine/xylasine IP

2)  Confirm the mouse is areflexic (by pinching hind paw) and breathing slowed to 50% of normal rate.

3)  Transcardially perfuse the mice with 10 ml of PBS followed by 10 ml of 4%PFA

4)  Remove brain and place in a tube with 4% PFA, store at 4C overnight.

5)  The next day, wash brains 3x 5 min with PBS

6)  After last wash, place brains in 30% sucrose in PBS for 2-3 days, until the brains

sink to the bottom of the tube

7)  Embed brains in TFM in a plastic mold and freeze using dry ice and 95% ethanol

slurry

8)  Store brains at -80C until use

On day of IHC experiment, section the desired brain regions using a cryostat, making 16-20um sections. Place sections on superfrost plus slides. Dry slides at 4C for at least 1 hour prior to starting the IHC.
 

Immunohistochemistry (IHC):

Day1:

Prepare the following solutions:

-  Antibody buffer – 100 mM L-lysine in PBS

-  Antibody dilution buffer – Antibody buffer + 5% Goat serum +0.3% Triton-x

-  Blocking buffer - Antibody buffer + 10% Goat serum + 0.3% Triton-x

-  PBS

-  PBS+0.2% Triton-X

1)  Take slides out of the 4C and let dry at RT for 5-10min

2)  During this time, prepare all the solutions needed (e.g., antibody buffer, blocking buffer)

3)  Apply the hydrophobic barrier pen to slides, drawing around the tissue sections.

4)  Once the barrier is dry, place slides in a humidified box and add blocking at ~200-250

ul/slide

5)  Incubate for 1 hour at RT in a humidified box

6)  During blocking prepare primary antibody diluted in antibody dilution buffer. Spin down

antibody dilutions at 13000rpm for 5 minutes before use.

7)  Remove blocking buffer and discard. Carefully dry the barrier and apply Primary Antibody diluted in antibody dilution buffer at - ~200-250ul per slide. Incubate overnight at 4C in a humidified box. Antibody dilutions are experimentally determined.

Day2:

8)  Remove antibody and place slides in a staining jar

9)  Wash 3X5 min with PBS+0.2% triton

10) During wash, prepare Secondary Antibody diluted in antibody dilution buffer. Spin down antibody dilutions at 13000rpm for 5 minutes before use

11)  After last wash, place slides back in the humidified chamber, carefully wipe any remaining liquid and check to ensure the hydrophobic barrier is intact

12)  Apply Secondary Antibody- ~200-250 ul/slide. Incubate for 2 hours at RT. Typical dilution 1:500 (experimentally determined)

13)  Wash 3X 5 min with PBS +0.2% triton

14)  Mount in DAPI-containing mounting medium, e.g. Slowfade gold. ~10ul per section. Apply

mounting media directly on each section.

15)  Carefully lower appropriately sized (40-60 length, 22-24 width, 1.5 thickness) coverslip,

remove excess liquid around edges and seal with clear nail polish

16)  Let dry for ~30min, away from light. Image soon after or store at 4C short term or -20C long

term.
 

Materials:

-  Superfrost plus slides (Fisher)

-  ImmEdge Hydrophobic Barrier Pen (Vector laboratories)

-  Staining jar (Fisher)

-  Coverslips (Fisher)

-  Mounting medium slowfade gold (Invitrogen)