Protocol required for Immunofluorescence in Bone Paraffin sections

Anatomy & Physiology Cell Anatomy / Physiology
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Hi, Does someone have a protocol about immunofluorescence in bone paraffin-embedded sections? Especially, I would like to know use which method for antigen retrieval?

Thanks

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Immunofluorescence (IF) on paraffin-embedded bone sections can be challenging due to the hard nature of the tissue and the need for effective antigen retrieval. Important Points to Remember are

  • Decalcification: Ensure that bone tissue is adequately decalcified before paraffin embedding. Over-decalcification can damage antigenicity, while insufficient decalcification can make sectioning difficult.
  • Control Sections: Include negative controls (no primary antibody) to assess background staining.
  • Optimization: Antibody concentrations and retrieval conditions may require optimization based on the specific antigen and tissue.

Here is a detailed protocol tailored for bone tissue:

Materials

  • Paraffin-embedded bone sections (cut at 5-7 µm)
  • Xylene, ethanol series (100%, 95%, 70%), and distilled water
  • Phosphate-buffered saline (PBS)
  • Heat-induced antigen retrieval (HIER) solution (e.g., Citrate buffer, pH 6.0 or Tris-EDTA buffer, pH 9.0)
  • Blocking solution (5% BSA or normal serum in PBS)
  • Primary antibody (specific to target antigen)
  • Secondary antibody (fluorophore-conjugated)
  • Mounting medium with DAPI or another nuclear stain
  • Coverslips
  • Fluorescence microscope

Protocol

1. Deparaffinization and Rehydration

  1. Deparaffinization:
  • Place slides in a series of xylene baths:
    • Xylene 1: 5 minutes
    • Xylene 2: 5 minutes
    • Xylene 3: 5 minutes
  1. Rehydration:
  • Immerse slides in a series of ethanol baths to rehydrate:
    • 100% ethanol: 5 minutes
    • 95% ethanol: 5 minutes
    • 70% ethanol: 5 minutes
  • Rinse slides in distilled water for 5 minutes.

2. Antigen Retrieval (Critical Step)

Method: Heat-Induced Epitope Retrieval (HIER) is the preferred method for bone tissues to unmask epitopes that are cross-linked during formalin fixation. Choose one of the following buffers based on the target antigen:

  • Citrate Buffer (pH 6.0): For most proteins.
  • Tris-EDTA Buffer (pH 9.0): For antigens that require more stringent conditions.

Procedure:

  1. Preheat the antigen retrieval buffer in a microwave, water bath, or pressure cooker.
  2. Place slides in a slide rack and immerse in the preheated buffer.
  3. Heat the slides:
  • Microwave: 5-10 minutes at 90-100°C.
  • Water bath: 20 minutes at 95°C.
  • Pressure cooker: 3-5 minutes at full pressure.
  1. Allow slides to cool at room temperature in the buffer for 20-30 minutes.
  2. Rinse slides in PBS to remove residual buffer.

3. Blocking

  1. Incubate slides in blocking solution (5% BSA or 10% normal serum in PBS) for 1 hour at room temperature to reduce non-specific binding.

4. Primary Antibody Incubation

  1. Dilute the primary antibody in PBS with 1% BSA.
  2. Apply the antibody to the sections and incubate overnight at 4°C in a humidified chamber.

5. Washing

  1. Wash slides in PBS three times for 5 minutes each to remove unbound primary antibody.

6. Secondary Antibody Incubation

  1. Dilute the fluorophore-conjugated secondary antibody in PBS with 1% BSA.
  2. Apply the secondary antibody and incubate for 1 hour at room temperature in the dark to protect from light.

7. Washing

  1. Wash slides in PBS three times for 5 minutes each in the dark.

8. Counterstaining (Optional)

  1. Apply a mounting medium with DAPI or another nuclear stain.
  2. Place a coverslip over the section and allow the medium to set.
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