Tissue histology and immunofluorescence

Anti-acetylated tubulin IF – Nerve IF

Kaylee Wells April 2020

Day 1

Primary Antibody Solution:

Anti-acetylated tubulin, T7451 mouse monoclonal (Located in small -20 ℃)

1:1500 dilution in 0.5% BSA (BSA made in PBST)

• PBS x2 (10 min)
• Pap pen around the sections and let dry ( >1 hour)
• PBS (10 min)
• 0.5% Trition X in PBST (5 min)
• Block in 0.5% BSA in PBST (>2 hours)
• Incubate with primary antibody (overnight) at 4 ℃ in humidification chamber

Day 2

Secondary Antibody Solution:

Goat-anti-mouse 488

A11001 – IgG Alexafluor 488 conj. 

Located in 2AB box in the 4 ℃ Walk-In

1:200 dilution in 0.5% BSA (BSA made in PBST)

Rhodamine Phalloidin:

REF: R415

Located in small -20 ℃

1:40 dilution in 1%BSA (BSA made in PBS)

DAPI:

1:1000 dilution in PBST

• PBST x3 (5 min)
• 0.5% BSA in PBST (20 min)
• Incubate with secondary antibody (2hours) at 4 ℃ in humidification chamber
• PBS x3 (3 min)
• Optional for muscle co-stain
  • Block in 1% BSA in PBS (30min)
  • Incubate with phalloidin (20 min) @RT
  • Wash with PBST x3 (5min)
• Stain with DAPI (10 min)
• PBST x3 (5min)
• Dry and use vectashild to coverslip
• Use clear nail polish to seal the edges.
• Store at 4 ℃
• Image within 3 days. 1 week max….