IMMUNOFLUORESCENCE ASSAY LAB CHECKLIST

I. Preparation of Reagents

Verify expiration dates of all reagents.

Check each reagent's label.
Note expiration dates.
Remove expired reagents from the workspace.
Document any issues with reagents.

Prepare and label primary antibodies.

Select appropriate primary antibodies.
Dilute antibodies as per protocol.
Label containers with antibody name and dilution.
Keep on ice until use.

Prepare and label secondary antibodies.

Choose suitable secondary antibodies.
Dilute according to the manufacturer's instructions.
Label containers clearly with antibody details.
Store in a cool place until needed.

Prepare blocking solution.

Select blocking agent (e.g., BSA, serum).
Dilute blocking agent in appropriate buffer.
Mix thoroughly to ensure homogeneity.
Label with concentration and date.

Prepare mounting medium.

Select appropriate mounting medium.
Mix components as per protocol.
Label with date and contents.
Store away from light until use.

Here are some additional steps that could be included in the I. Preparation of Reagents section of your immunofluorescence assay lab checklist

Ensure all reagents are at the appropriate temperature before use

Check refrigeration temperature.
Allow reagents to equilibrate to room temperature.
Avoid repeated freeze-thaw cycles.
Prepare a cooling station if needed.

Prepare dilution buffers (e.g., PBS, Tris) as needed for antibody dilution

Choose required buffer type.
Prepare buffer according to recipe.
pH check and adjust if necessary.
Label with buffer type and date.

Check and prepare any necessary controls (e.g., isotype controls, positive/negative controls)

Identify required control antibodies.
Prepare controls at the same dilution as test samples.
Label controls clearly.
Keep controls organized for easy access.

Prepare any additional reagents required for specific staining protocols (e.g., counterstains, enzyme substrates)

Review protocol for additional reagents.
Prepare each reagent according to specifications.
Label all additional reagents with details.
Store under recommended conditions.

Thoroughly mix reagents before use to ensure consistency

Use vortex mixer for solutions.
Gently invert delicate reagents.
Avoid bubbles in sensitive solutions.
Ensure homogeneity before transferring.

Store prepared reagents at the appropriate temperature and conditions until needed

Place reagents in designated storage.
Check temperature settings regularly.
Label all storage containers clearly.
Document storage conditions.

Document the preparation of reagents, including concentrations and volumes used

Create a reagent log.
Record volumes, concentrations, and dates.
Include names of preparers.
Store log in an accessible location.

Dispose of any expired or unused reagents according to safety protocols

Identify expired reagents for disposal.
Follow institutional safety protocols.
Label waste containers accordingly.
Document disposal actions in the log.

II. Sample Preparation

Collect samples according to protocol.

Fix samples using appropriate fixative.

Permeabilize samples if necessary.

Wash samples with buffer solution.

III. Staining Procedure

Apply blocking solution to samples and incubate.

Dilute primary antibody to recommended concentration.

Apply primary antibody to samples and incubate.

Wash samples to remove unbound primary antibody.

Dilute secondary antibody and apply to samples.

Incubate with secondary antibody and wash.

IV. Microscopy Setup

Ensure microscope is clean and functional.

Set appropriate filters for fluorescent detection.

Calibrate light intensity and exposure time.

Prepare slides for imaging.

V. Data Acquisition

Capture images of stained samples.

Record settings used for each image.

Save images in appropriate format and location.

VI. Data Analysis

Analyze images using software.

Quantify fluorescence intensity if applicable.

Generate and save data reports.

VII. Cleanup and Disposal

Dispose of hazardous materials according to guidelines.

Clean work surfaces and equipment.

Store reagents properly for future use.

VIII. Documentation

Complete lab notebook with all procedures and results.

Update inventory of reagents used.

Archive images and data in designated folders.