1. Immunostaining 5μm Tissue Sections for Confocal Imaging
By: Ryan King

2. Properly Labelling the Slide is Critical for Record Keeping
Date
Initials
Sample Name
Tissue Prep
(i.e. Fresh Frozen)
Make sure to include the Date, Your name or initials, the sample name and how the tissue was prepared on every slide.

3. If putting two samples on one slide leave ample room between samples
Date
Initials
Sample Name A B
If putting two samples on one slide leave ample room between samples
Tissue Prep
(i.e. Fresh Frozen)
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)

4. Immunostaining Outline
Day 1
Wash 1x5min in PBS
Fix 1x5min in 2%PFA
Wash 3x5min in PBS
Block 1-2 hours in 1% BSA
Incubate with 1o antibody overnight

5. 1 x 5 Min wash in PBS
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Pour 1x PBS directly into slide holder, enough to cover slide up to the label (~40mL)
Place slide holder on shaker and set timer for 5 minutes
Pour 1x PBS into PBS waste container

6. MOVE SLIDES UNDER SNORKEL OR INTO FUME HOOD
1 x 5 Min wash in PFA
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
MOVE SLIDES UNDER SNORKEL OR INTO FUME HOOD
Pour 2% PFA directly into slide holder, enough to cover slide up to the label (~40mL)
Place slide holder on shaker and set timer for 5 minutes (Keep under snorkel)
Pour 2% PFA into PFA waste container

7. 3 x 5 Min wash in PBS
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Pour 1x PBS directly into slide holder, enough to cover slide up to the label (~40mL)
Place slide holder on shaker and set timer for 5 minutes
Pour 1x PBS into PBS waste container
Repeat 3 times

8. Block in 1x BSA
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Vacuum excess PBS off of slides being careful to not vacuum directly over sample
Use PAP pen to draw a hydrophobic circle around your sample, careful not to apply hydrophobic circle onto sample
Allow hydrophobic layer to dry completely
Apply ~100μL of BSA onto each sample
Move samples onto sponge and cover to keep out of the light
Let slides block at room temperature for 1-2 hours.

9. Application of primary antibody
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Vacuum off BSA from samples, careful not to vacuum off the sample itself
Apply ~30μL of your desired primary antibody* to the sample
Move samples back onto sponge and cover to keep out of the light
Place samples in 4oC to store overnight (~24 hours)
Prior to application of the antibody the antibody should be diluted into BSA.
= Primary Antibody

10. Wash in PBS A B Uncover samples and remove from sponge
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Uncover samples and remove from sponge
Perform one rinse with PBS
Perform 4 five minute washes with PBS in slide holder
Prior to application of the antibody the antibody should be diluted into BSA.
= Primary Antibody

11. Application of secondary antibody
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Apply ~30μL of your desired secondary antibody* to the sample
Move samples back onto sponge and cover to keep out of the light
Prior to application of the antibody the antibody should be diluted into BSA.
= Primary Antibody
= Secondary Antibody

12. Wash in PBS and Nuclear Stain
Date
Initials
Sample Name A B
Tissue Prep
(i.e. Fresh Frozen)
Remove from cover and rinse with PBS
Perform three five minute washes with PBS in slide holder
Perform one five minute wash with PBS + Hoechst nuclear stain (diluted 1:20,000) in the slide holder
Prior to application of the antibody the antibody should be diluted into BSA.
= Primary Antibody
= Secondary Antibody