IF

Immunofluorescence (IF) technique combines the immunological method (specific binding of antigen and antibody) with fluorescence labeling method. The fluorescence produced by fluorescein could be detected with the fluorescence microscope, thus IF can be used in the localization analysis of specific antigens in cells.

Here Elabscience lists the common Immunofluorescence troubleshooting.

1. Weak fluorescence signals or no fluorescence expression

2. High background of fluorescence

3. Fast fluorescence quenching

4. Cell auto-fluorescence

Notes for fluorescent double staining

For Indirect method:

1) It is recommended to use primary antibodies from two different species, and secondary antibodies with different fluorescence labeling.

2) It is recommended to incubate one primary antibody, then incubate the fluorescence secondary antibody which matches the primary antibody, then followed with incubation of another primary antibody and its matched fluorescence secondary antibody.

3) Set positive control and negative control.

4) Use blocking serum that was collected from the species in which the secondary antibody was raised.

The other procedures are the same as conventional IF experiments.

For Direct method:

The species of primary antibodies are not specially required, and the fluorescent labels of two primary antibodies should be different.

Immuno Fluorence Staining Kits are developed for immunofluorescence detection of cell or tissue sections. When there is an appropriate antibody to detect specific target protein, fluorescence can be detected by immunofluorescence staining kit.

Recommend ELabscience® Immuno Fluorescence Staining Kits

Experimental Procedure

1. Preparation of Immunofluorescence Staining

A.Preparation of Fixation Solution

It is recommended to use 4% Paraformaldehyde as the fixation solution (E-IR-R113), or use ethanol,methanol or other types of fixative according to specific primary antibody or sample.

B.Preparation of TBST Working Buffer

It is recommended to use TBST as washing Buffer. Use Elabscience ® 10× TBST (E-BC-R335) and dilute to 1 ×TBST Working Buffer with deionized water at ratio of 1:9.

C.Preparation of Antibody Dilution Solution

It is recommended to use Elabscience ® Antibody Dilution Buffer (E-IR-R106) or PBS as primary antibody dilution Buffer.

D.Dilute Primary Antibody

Dilute the primary antibody according to the manual of primary antibody.

E.Dilute the Secondary Antibody

It is recommended to use Elabscience ® Antibody Dilution Buffer (E-IR-R106) or PBS as secondary antibody dilution Buffer. Dilute the secondary antibody with antibody dilution Buffer at the dilution of 1:50. The dilution ratio can be increased or decreased appropriately according to the intensity of fluorescence.

2. Immuno Fluorescence Staining for Adherent Cells

• A.Immerse a clean cover glass into 70% ethanol for 5 min or a longer time, dry it in the sterile super clean table or wash it with cell culture grade PBS or 0.9% NaCl solution for 3 times, then wash it with cell culture solution. Place the cover glass into six hole plate, seed the cells on the glass overnight to make it about 50% – 80% full.
• B.Treat the cells according to the specific experimental purpose, then discard the culture solution, add 1 ml fixation solution, and incubate for 10 min or a longer time (or overnight at 4°C).
• C.Discard the fixation solution, wash the glass with TBST working Buffer for 3~5 min, 3 times. Discard the liquid.
• D.Blocking. Add 100 μL Normal Goat Serum (Ready-to-Use) (E-IR-R110) to each glass and incubate for 30 min.
• E.Discard the blocking Buffer, add 100 μL diluted primary antibody and incubate at 37°C in wet box for 60 min (Or incubate at 4°C overnight).
• F.Discard the primary antibody, wash the glass with TBST Working Buffer for 3~5 min, 3~5 times.
• G.Discard the liquid. Add 100 μL diluted secondary antibody and incubate at 37°C in wet box for 60 min.
• H.Wash the glass with TBST Working Buffer for 3~5 min, 3~5 times, avoid light during the washing.
• I.Nuclear staining. Add DAPI Reagent (E-IR-R103) and incubate in wet box for 5 min, wash the glass with TBST Working Buffer for 5 min, wash for 4 times to remove the redundant DAPI Reagent.
• J.Add one drop of Anti-Fluorescence Quenching Agent (E-IR-R119) on a slice glass, cover the glass with cells, avoid air bubbles. Make the cell contact with the Anti-Fluorescence Quenching Agent, do not reverse it.
• K.Observe the result by fluorescence microscope.

3. Immuno Fluorescence Staining for Suspension Cells

• A.Collect the cells by centrifugation into a 1.5 ml centrifuge tube. Scatter the cells gently after discarding the supernatant.A.Collect the cells by centrifugation into a 1.5 ml centrifuge tube. Scatter the cells gently after discarding the supernatant.
• B.Add 0.5 ml fixation solution to re-suspend the cells gently, and incubate for 10 min or a longer time (or overnight at 4°C).
• C.Centrifuge the cells and discard the fixation solution, wash the cells with TBST Working Buffer for 3~5 min, 3 times.
• D.Discard the TBST working Buffer and left about 50 μL liquid for the last time, re-suspend the cells, add the cells to a clean slide glass, make sure that the cells are uniform distributed.
• E.Slightly dry the cells to make the cells are attached to the slide and not easy to flow with the liquid. If the conditions permit, the cells can be attached to the slide by centrifugation using a suitable centrifuge.
• F.Blocking. Add 100 μL Normal Goat Serum (Ready-to-Use) (E-IR-R110) to each glass and incubate for 30 min.
• G.Discard the blocking Buffer, add 100 μL diluted primary antibody and incubate at 37°C in wet box for 60 min (Or incubate at 4°C overnight).
• H.Discard the primary antibody, wash the glass with TBST working Buffer for 3~5 min, 3~5 times.
• I.Discard the liquid. Add 100 μL diluted secondary antibody and incubate at 37°C in wet box for 60 min.
• J.Wash the glass with TBST Working Buffer for 3~5 min, 3~5 times, avoid light during the washing.
• K.Nuclear staining. Add DAPI Reagent (E-IR-R103) and incubate in wet box for 5 min, wash the glass with TBST Working Buffer for 5 min, wash for 4 times to remove the redundant DAPI Reagent.
• L.Add one drop of Anti-Fluorescence Quenching Agent (E-IR-R119) on a slice glass, cover the glass with cells, avoid air bubbles. Make the cell contact with the Anti-Fluorescence Quenching Agent, do not reverse it.
• M.Observe the result by fluorescence microscope.

4. Immuno Fluorescence Staining for Tissue Slice

• A.For paraffin section, dewaxing, hydration and antigen repair should be completed first. For frozen sections, follow the steps below.A.For paraffin section, dewaxing, hydration and antigen repair should be completed first. For frozen sections, follow the steps below.
• B.Incubate with fixation solution for 10 min or a longer time (or overnight at 4°C).
• C.Discard the fixation solution, wash the slice with TBST Working Buffer for 3~5 min, 3 times. Discard the liquid.
• D.Blocking. Add 100 μL Normal Goat Serum (Ready-to-Use)(E-IR-R110) to each glass and incubate for 30 min.
• E.Discard the blocking Buffer, add 100 μL diluted primary antibody and incubate at 37°C in wet box for 60 min (Or incubate at 4°C overnight).
• F.Discard the primary antibody, wash the glass with TBST Working Buffer for 3~5 min, 3~5 times.
• G.Discard the liquid. Add 100 μL diluted secondary antibody and incubate at 37°C in wet box for 60 min.
• H.Wash the glass with TBST Working Buffer for 3~5 min, 3~5 times, avoid light during the washing.
• I.Nuclear staining. Add DAPI Reagent (E-IR-R103) and incubate in wet box for 5 min, wash the glass with TBST Working Buffer for 5 min, wash for 4 times to remove the redundant DAPI Reagent.
• J.Add one drop of Anti-Fluorescence Quenching Agent (E-IR-R119) on a slide glass, cover the glass with cells, avoid air bubbles. Make the cell contact with the Anti-Fluorescence Quenching Agent, do not reverse it.
• K.Observe the result by fluorescence microscope.