Immunofluorescence on frozen sections

Tissue preparation

  1. Snap-freeze tissue in isopentan precooled close to its freezing point in liquid nitrogen
  1. Cut sections 5 microns thick and mount onto poly-L-lysine coated glass slides**
  1. Air-dry sections under a fan for 2-24 hours

Preparation of Poly-L-Lysine coated slides

Use only: Poly-L-Lysine, Hydrobromide (Sigma, Product No: P1524)

Preparation of 600 slides:

50 ug/ml in 10 mM Tris buffer ph 7.4 (total 1.5 L)

Solution may be used twice.

Coating of slides:

  1. Clean slides for 24 hours in detergent (Extran MA O1, E. Merck, Article-No: 1.07555) (Stability 2 weeks)
  2. Clean slides for 30 min. in hot water, then for three hours in cold water
  3. Clean slides in demineralized water
  4. Put slides for 10 min. in Poly-L-Lysine
  5. Dry slides at room temperature, store at room temperature in the original boxes (dust free)

*Alternative: Superfrost Plus Gold Slides (very expensive!)

(Menzel; Art. No: K 5800AMNZ72)

Links

Sigma-Aldrich Co.:

Merck KgaG (D-Darmstadt):

Menzel (D-38116 Braunschweig,Saarbrücker Strasse 248):

Direct Immunofluorescence staining

  1. Sections are thawed at room temperature and dried for 30 minutes
  1. Wash slides 5 min. with cold PBS-Tween20 (0.1%) at room temperature
  1. Remove excess PBS-Tween
  1. Add 100µl diluted antibody (see separate list of antibodies) and incubate in a moist chamber for 30 min. at room temperature
  1. Remove excess antiserum by PBS
  1. Wash slides in cold PBS for 5 min at room temperature (2x)
  1. Mount with fluorescence mounting medium (Dako, Code S3023) and cover with coverslip
  1. Seal coverslip with nail polish (sealing-fluid)

Link

Dako (DK-2600 Glostrup):

Indirect Immunofluorescence staining

  1. Sections are thawed at room temperature and dried for 30 minutes
  1. Wash slides 5 min. with cold PBS-Tween20 (0.1%) at room temperature
  1. Remove excess PBS-Tween
  1. Add 100µl diluted primary antibody (see separate list of antibodies for anti-human C4d (Quidel A213) and anti-human C5-9 (Quidel A239)) and incubate in a moist chamber for 30 min. at room temperature
  1. Remove excess antiserum by PBS
  1. Wash slides in cold PBS for 5 min at room temperature (2x)
  1. Add 100µl diluted Goat anti-mouse IgG(H+L)-Alexa Fluor488 (Molecular Probes, A-11029) as second antibody and incubate in a moist chamber for 30 min. at room temperature
  1. Remove excess antiserum by PBS
  1. Wash slides in cold PBS for 5 min at room temperature (2x)
  1. Mount with fluorescence mounting medium (DAKO, S3023,) and cover with coverslip
  1. Seal Coverslip with nail polish (sealing-fluid)

Links

Quidel Corporation:

Dako (DK-2600 Glostrup):

Molecular Probes: