Immunofluorescence Assay (IFA)

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IFA Calculator

Immunofluorescence Assay  ·  Sindbis · Influenza · YFV · Seneca ⏱️ Timers ready Antibody dilutions

⏱️ IFA Protocol click Start on any step

⏳ Timer ready 00:00

1 Fixation: 3.7% formaldehyde (10 min) OR 100% methanol (10 min) OR 80% methanol (20 min) at 4°C. 10-20 min

2 Wash: 2-3× with PBS. 100-200 µL per well (plate dependent). 5 min

3 Permeabilization: 0.2% Triton X-100 for 8-10 min. 10 min

4 Wash: 3× with PBS. 5 min

5 Blocking: 1% PBS-BSA, 30 min at RT. 30 min

6 Wash: 2-3× with PBS. 5 min

7 Primary Antibody: Diluted in 1% PBS-BSA. Incubate 1 hour at 37°C 60 min

8 Wash: 5-6× with PBS (100-200 µL/well). 10 min

9 Secondary Antibody: Anti-species IgG (1:1000) in 1% PBS-BSA. 30-60 min at RT/37°C. PROTECT FROM LIGHT! 60 min

10 Wash: 5-6× with PBS. Protect from light. 10 min

11 Optional DAPI: 1:2000 in PBS for nuclear staining. 5 min

12 Visualization: Examine under fluorescence microscope. Store in dark at 4°C in PBS. — no timer

⚠️ Critical: Protect fluorophore-conjugated antibodies from light! Wrap tubes in foil. Keep samples in dark during and after staining.

▼ REAGENT CALCULATOR — prepare before starting ▼

🧪 1. Plate Format & Samples adjust volumes

Plate format

96-well plate 48-well plate 24-well plate 12-well plate 6-well plate Custom 40-100 µL/well

Custom volume per well (µL)

µL

Number of wells (samples + controls)

wells

Overage (extra % for pipetting)

%

📋 Plate reference volumes:

Plate	Fix/Wash (µL)	Antibody (µL)	Triton (µL)	Storage (µL)
96-well	100-200	40-100	40-100	100-200
48-well	200-300	100-200	100-200	200-300
24-well	300-500	150-250	150-250	300-500
12-well	500-1000	250-500	250	500-1000
6-well	1000-2000	500-1000	500	1000-2000

Total wells

—

including overage

Volume per well

— µL

antibody/reagent volume

Reagent	Per Well (µL)	Total (µL)

💡 PBS wash volumes can be adjusted based on plate format. Protect fluorophores from light!

🧫 2. Antibody Dilutions primary · secondary

Virus / Target

Sindbis (Mouse ascitic fluid, 1:500) Influenza A (Rabbit, 1:500 or 1:1000) Yellow Fever (Rabbit NS4B, 1:500) Seneca Valley (Mouse Mab, 1:30,000; Rabbit 1:500) Custom antibody

Primary antibody dilution (1:X)

1 : mouse ascitic fluid

Seneca special dilution (1:30 first, then 1:1000)

Intermediate 1:30 then 1: total 1:30,000

Secondary antibody dilution (1:X, anti-species IgG)

1 : anti-Mouse IgG anti-Rabbit IgG anti-Goat IgG anti-Human IgG

DAPI dilution (optional, 1:X for nuclear stain)

1 : optional

Primary Ab total volume

— µL

neat + diluent

Secondary Ab total volume

— µL

neat + diluent

Antibody	Neat Ab (µL)	Diluent (1% PBS-BSA) (µL)	Total (µL)

🌑 Light-sensitive! Prepare dilutions in foil-wrapped tubes. Keep samples in dark during and after staining.

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