Detailed BrdU Immuno Protocol with anti-YFP pre-staining

As in Christenson Wick, Tetzlaff, & Krook-Magnuson, eLife (2019) paper

Performed on free-floating 50μm sections in 24-well plates.

Use gentle shaker for all steps.

See tables below for in-house solutions.

Ensure Institutional approval and refer to Material Safety Data Sheets for safe handling of all hazardous materials (e.g. paraformaldehyde, picric acid, hydrochloric acid, and BrdU)

DAY 1

Rinse 50μm sections 3x for 5 mins each in 0.1M PB at room temperature (RT)

Rinse sections 2x for 10 mins each in TBS at RT

Incubate in anti-YFP primary antibody solution overnight at 4°C

To make 1mL of anti-YFP 1° antibody solution: need 0.25mL per well + a little extra

0.5mL - TBS

0.5mL - TBS + 0.5% Triton

1μL - goat anti-GFP (1:1000; Abcam ab6673)

DAY 2

Rinse sections 3x 5 mins each in TBS at RT

Incubate in secondary antibody solution for 2 hrs at RT

To make 1mL of 2° antibody solution: need 0.25mL per well + a little extra

1mL - TBS

2μL - Alexa Fluor 488 donkey anti-goat (1:500; Abcam ab150129)

Rinse sections 3x 5 mins each in 0.1M PB at RT

Incubate in 4% paraformaldehyde fixative overnight at 4°C

DAY 3

Rinse sections 3x 5 mins each in 0.1M PB at RT

Denature DNA by incubating sections in 1M HCl for 30 min at 45°C

Follow appropriate safety precautions when handling HCl.

Rinse sections 3x 5 mins each in 0.1M PB at RT

Incubate in 1:1 ratio of TBS+0.5% Triton and 10% normal goat serum (NGS) for 1 hr at RT

Incubate in BrdU primary antibody solution for 48 hours at 4°C

To make 1mL of 1° antibody solution: need 0.25mL per well + a little extra

0.5mL - TBS+0.5% Triton

0.5mL - 10% NGS (Invitrogen 50062Z)

1μL - rabbit anti-BrdU (1:1000; ThermoFisher PA5-32256)

DAY 4 - nothing

DAY 5

Rinse sections 3x 5 mins each in 0.1M PB at RT

Incubate in secondary antibody solution for 2 hrs at RT

To make 1mL of 2° antibody solution: need 0.25mL per well + a little extra

800μL - TBS

200μL - 10% NGS

2μL - Alexa Fluor 594 goat anti-rabbit (1:500)

Rinse sections 3x 5 mins each in 0.1M PB at RT

Mount sections with mounting media with DAPI (Vectashield Antifade Mounting Media with DAPI, H-1200)

Ingredients for our Lab-made Solutions:

0.2M PB (Stock solution, store in fridge) -- dilute to 0.1M PB as needed

Ingredient	Amount
H₂O	2L
Na₂HPO₄	45.44 g
NaH₂PO₄	9.3 g

4% PFA (store in fridge) -- makes 25mL. Can be scaled up as needed.

Caution: follow safety precautions as per Material Safety Data Sheets for handling paraformaldehyde & picric acid.

Ingredient	Amount
0.2M PB	12.5mL
10% Paraformaldehyde	10mL
H₂O	2.5mL
Picric Acid	25µL

TBS (store in fridge)

Ingredient	Amount
H₂O	2L
Trizma HCl	13.22g
Trizma Base	1.90g
NaCl	18.0g