May 07, 2025

Public workspaceImmunofluorescence Staining of Bacterial Samples V.3

DOI
dx.doi.org/10.17504/protocols.io.4r3l2eknjl1y/v3
  • 1INESC-MN;
  • 2Instituto Superior Técnico, Universidade de Lisboa;
  • 3Unidade Militar Laboratorial de Defesa Biológica e Química, Exército Português;
  • 4Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.
  • Advanced Integrated Microsystems Doctoral Program
Pedro Fonseca
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2eknjl1y/v3
Protocol Citation: Pedro Fonseca 2025. Immunofluorescence Staining of Bacterial Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2eknjl1y/v3Version created by Pedro Fonseca
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2025
Last Modified: May 07, 2025
Protocol Integer ID: 217916
Keywords: Immunofluorescence, Bacterial Staining, Fluorescence Microscopy, Antibody Labeling, Monolayer Preparation, Slide Coating
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Abstract
This protocol describes the preparation, fixation and immunostaining of bacterial samples for fluorescence microscopy. It includes detailed steps for monolayer preparation, blocking, primary and secondary antibody incubations, and mounting, with options to optimize slide preparation and antibody incubation for improved reproducibility and signal quality.
Guidelines
  • Always keep fluorescent antibodies and stained slides protected from light.
  • Keep antibodies on ice during preparation.
  • Use clean, particle-free solutions and sterile pipette tips.
  • For reproducibility, prefer pre-coated Poly-L-lysine slides when possible.
  • Pre-test antibody concentrations if using new lots or targets.
Materials
  • 70% Ethanol
  • Poly-L-lysine solution (1 mg/mL) or Pre-coated Poly-L-lysine slides
  • Phosphate Buffered Saline (PBS), pH 7.4
  • PBS with 0.5% Triton X-100 (PBS-Tx)
  • Fish Skin Gelatin 0.2% PBS solution (blocking agent)
  • Ultrapure water (preferably Milli-Q)
  • Primary antibodies (1:100 working solution; on ice)
  • Fluorescent secondary antibodies (1:100 working solution; on ice, protected from light)
  • 1.5 mL Eppendorf tubes
  • Microscope slides
  • P200 micropipette and sterile tips
  • Humid chamber
  • Slide holders for washing
  • DAKO pen
  • Filter paper
  • Coverslips
  • Mounting media (e.g., VectaShield® or Fluoromount™)
  • Clear nail varnish (optional)
Safety warnings
  • Handle all fluorescent reagents in low light to prevent signal degradation.
  • Avoid letting slides dry during staining steps.
  • Ensure thorough but gentle washes to prevent detaching bacterial monolayers.
  • Carefully apply and seal coverslips to prevent sample loss and preserve fluorescence.
Before start
  • Turn on the humid chamber to equilibrate at desired temperature.
  • Prepare all antibody working solutions fresh.
  • Clean glassware and ensure the workspace is free of dust and particles.
  • Have all washing buffers (PBS, PBS-Tx) ready and at room temperature.
Bacterial Preparation
Bacterial Preparation
Transfer 1 mL of 24-hour bacterial culture into a 1.5 mL Eppendorf tube.
Centrifuge at 4000 × g for 4 minutes.
Discard supernatant and resuspend pellet in 1 mL PBS.
Repeat centrifugation and resuspension two more times (total three washes).
After final wash, resuspend pellet in 100 µL PBS.
Slide Preparation
Slide Preparation
Option 1: Manual Poly-L-lysine Coating
Clean slides with 70% ethanol.
Pipette 100 µL Poly-L-lysine solution onto filter paper.
Firmly swipe the filter paper once across each slide.
Option 2: Pre-coated Slides
Use pre-coated Poly-L-lysine slides directly.
Draw hydrophobic barriers using the DAKO pen.
Pipette 100 µL bacterial suspension into compartments.
Incubate in a humid chamber at room temperature for 10–15 minutes.
Gently blot excess with a paper towel.
Check under microscope for monolayer formation.
Blocking
Blocking
Pipette 100 µL 0.2% Fish Skin Gelatin into each compartment.
Incubate in a humid chamber for 30 minutes at room temperature.
Wash slides:

  • Brief rinse with PBS-Tx

  • 5-minute wash with PBS
Primary Antibody Incubation
Primary Antibody Incubation
Pipette 30 µL primary antibody working solution into experimental compartments.
Pipette 30 µL Fish Skin Gelatin into control compartments.
Incubation options:

  • 1 hour at 37°C in humid chamber, or

  • Overnight at 4°C in humid chamber (recommended for improved sensitivity)
Wash slides:

  • Brief rinse with PBS-Tx

  • 10-minute PBS-Tx wash at room temperature

  • 5-minute PBS wash
Secondary Antibody Incubation
Secondary Antibody Incubation
Add 30 µL fluorescent secondary antibody to all compartments.
Incubate for 30 minutes at 37°C in the dark.
Wash slides:

  • Brief rinse with PBS-Tx

  • 10-minute PBS-Tx wash at room temperature

  • 5-minute PBS wash
Mounting
Mounting
Apply mounting medium (e.g., VectaShield® or Fluoromount™).
Gently place coverslip, avoiding air bubbles.
Optionally seal coverslip edges with clear nail varnish.
Store slides in the dark until imaging.
Visualize using a fluorescence microscope.
Protocol references
1. Skerker JM, Berg HC. Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci U S A. 2001;98(12):6901–4.

2. Kuru E, Hughes HV, Brown PJ, Hall E, Tekkam S, Cava F, et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew Chemie - Int Ed. 2012;51(50):12519–23.

3. Im K, Mareninov S, Diaz MFP, Yong WH. An Introduction to Performing Immunofluorescence Staining. In: Yong WH, editor. Physiology & behavior [Internet]. New York, NY: Springer New York; 2019. p. 299–311. (Methods in Molecular Biology; vol. 1897). Available from: http://link.springer.com/10.1007/978-1-4939-8935-5