Immunostaining 5μm Tissue Sections for Confocal Imaging By: Ryan King ryanking@vt.edu

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.

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)

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

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

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

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

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.

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

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

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

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 33342 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