docs/protocols/cell_staining_v1.yaml

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# Protocol metadata
id: PROT-0001
name: Cell Staining Protocol
version: 1.0
description: Protocol for immunofluorescence staining of cells
author: Alice Smith
created: 2025-05-05
last_updated: 2025-05-07
category: microscopy

# Materials required
materials:
  - name: Primary antibody (Anti-XYZ)
    dilution: 1:500
    storage: -20°C
  - name: DAPI nuclear stain
    concentration: 1 μg/mL
    storage: 4°C, protected from light
  - name: Paraformaldehyde (PFA)
    concentration: 4% in PBS
    storage: 4°C, prepare fresh if possible
  - name: PBS (Phosphate Buffered Saline)
    concentration: 1X
    storage: Room temperature
  - name: Mounting medium
    type: Anti-fade
    storage: 4°C

# Equipment required
equipment:
  - name: Fluorescence microscope
    settings: Appropriate filter sets for antibody fluorophores and DAPI
  - name: Coverslips/chamber slides
    type: Glass, tissue-culture treated
  - name: Fine-tip forceps
    use: Handling coverslips
  - name: Humidified chamber
    use: Antibody incubation

# Protocol steps
steps:
  - step: 1
    action: "Fix cells with 4% PFA"
    details: "Incubate for 10 minutes at room temperature"
  - step: 2
    action: "Wash 3x with PBS"
    details: "5 minutes per wash, gentle rocking"
  - step: 3
    action: "Permeabilize cells"
    details: "Use 0.1% Triton X-100 in PBS for 5 minutes at room temperature"
  - step: 4
    action: "Block non-specific binding"
    details: "Incubate with 3% BSA in PBS for 30 minutes at room temperature"
  - step: 5
    action: "Add primary antibody (Anti-XYZ)"
    details: "Dilute 1:500 in blocking solution, incubate for 1 hour at room temperature or overnight at 4°C"
  - step: 6
    action: "Wash 3x with PBS"
    details: "5 minutes per wash, gentle rocking"
  - step: 7
    action: "Add DAPI stain"
    details: "Incubate for 5 minutes at room temperature, protected from light"
  - step: 8
    action: "Wash 2x with PBS"
    details: "5 minutes per wash, gentle rocking"
  - step: 9
    action: "Mount and seal slides"
    details: "Apply mounting medium, place coverslip, and seal edges with nail polish if needed"
  - step: 10
    action: "Image slides"
    details: "Use appropriate filter sets and exposure times for each fluorophore"

# Critical parameters
critical_parameters:
  - parameter: "Antibody dilution"
    details: "Optimal dilution may vary by lot and application; validate before use"
  - parameter: "Fixation time"
    details: "Over-fixation can mask epitopes; under-fixation can compromise cell morphology"
  - parameter: "Light exposure"
    details: "Minimize exposure to light after fluorophore addition to prevent photobleaching"

# Troubleshooting
troubleshooting:
  - problem: "High background signal"
    solution: "Increase blocking time, increase wash times, or reduce antibody concentration"
  - problem: "Weak or no signal"
    solution: "Check antibody viability, increase concentration, or optimize fixation method"
  - problem: "Non-specific staining"
    solution: "Increase blocking time/concentration, validate antibody specificity, add serum from secondary host"

# Safety considerations
safety:
  ppe: "Lab coat, gloves, and eye protection required"
  hazards: "Paraformaldehyde is toxic; work in fume hood and dispose of waste properly"

# Quality control
quality_control:
  - check: "Include negative control (no primary antibody)"
    criteria: "Should show minimal background fluorescence"
  - check: "Include positive control"
    criteria: "Sample known to express target protein"

# References
references:
  - "Donaldson JG. (2015) Immunofluorescence staining. Curr Protoc Cell Biol. 69:4.3.1-4.3.7"

# Notes
notes: |
  - Ensure all reagents are at room temperature before starting.
  - Use appropriate personal protective equipment (PPE) when handling chemicals.
  - Adjust antibody dilution based on specific cell type and experimental conditions.
  - Store unused antibodies at -20°C for long-term storage.
  - Document any deviations from the protocol in the lab notebook.
  - Always include a control sample for comparison.
  - Dispose of all waste according to local regulations.
  - Ensure that the microscope is calibrated and functioning properly before imaging.
  - Record all imaging parameters for reproducibility.
  - If using a different staining protocol, ensure compatibility with the antibodies used.
  - For best results, use fresh reagents and avoid freeze-thaw cycles.
  - If using multiple antibodies, ensure they are compatible and do not cross-react.
  - If using a secondary antibody, ensure it is compatible with the primary antibody and the detection method.
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