How can I avoid uneven IHC staining on free floating tissue from frozen human CNS sections (cryostat 50um thickness)?

Hello Paulina, when you are using free floating tissue sections don't use AR.

AR is recommanded when you use paraffin sections. During the AR the tissue swells most the time. If the concentration of the primary ab is too hight this could also result in a patchy labeling. When you are doing peroxidase inactivation prepare a solution of 0.3% H2O2 in PBS no EtOH. Incubation time 20 -30 min, folled by 3x10 min rinsing in PBS. increase all washing steps from 3 x5 to 3 x 10 min. Instead of NFDM you can use normal serum. And add 0.2 to 0.5% TritonX100 in all of your dilution media. Make different dilutions of the primary ab to find out which is the bestone. 1:500 up to 1:1000 is recommandetd by SySy. You can also increase the incubation time fom 3 to 5 day, but you can start at first with a over night incubation. You can enhance your result by adding Ni-ammoniumsulfate in the DAB solution or if this is not enaough you can use the biotylated Tytramide enhancement technique. Hope these recommandations will help you to get better results. Good luck!

Idowu Goodnews Oluwaseyi

Achieving even immunohistochemistry (IHC) staining on free-floating tissue sections, particularly from delicate and variable tissues like frozen human central nervous system (CNS) sections, requires careful handling and optimized protocols. Here are several strategies to help avoid uneven staining:

Preparation and Handling

Sectioning:Ensure sections are cut uniformly at 50 μm using a well-calibrated cryostat. Handle sections gently to avoid damage and tearing.

Storage:Store sections properly if not used immediately. Free-floating sections can be stored in cryoprotectant solutions at -20°C or in PBS with 0.1% sodium azide at 4°C.

Washing and Blocking

Washing:Thoroughly wash sections in PBS or Tris-buffered saline (TBS) to remove freezing medium and other debris. Perform multiple washes (at least 3 times, 5 minutes each) to ensure complete removal of any residual freezing medium.

Permeabilization and Blocking:Permeabilize sections with 0.1-0.5% Triton X-100 in PBS/TBS if your antibody requires access to intracellular targets. Block non-specific binding sites using 5-10% normal serum (from the species in which the secondary antibody was raised) or 5% BSA for at least 1 hour at room temperature or overnight at 4°C.

Antibody Incubation

Primary Antibody Incubation:Incubate sections with the primary antibody diluted in blocking solution. Gentle agitation during incubation can help ensure even exposure of the tissue to the antibody. Perform primary antibody incubation overnight at 4°C for better penetration and binding.

Secondary Antibody Incubation:After thorough washing, incubate sections with the secondary antibody, also diluted in blocking solution, with gentle agitation.

Staining Procedures

Washing Steps:Ensure thorough washing between primary and secondary antibody incubations to reduce background and non-specific binding. Use a gentle rocking platform to avoid tearing delicate tissue sections.

Avoiding Air Bubbles:Carefully remove any air bubbles that may form on the tissue during incubation steps, as these can cause uneven staining. Use fine brushes or pipette tips to gently nudge bubbles away from the sections.

Mounting

Mounting Sections:Carefully transfer sections to slides using fine brushes or pipettes to avoid folds or overlaps. Remove excess mounting medium gently and ensure the sections lay flat without creases.

General Tips

Consistent Protocols:Ensure consistency in timing, temperature, and agitation during all steps of the IHC protocol. Use appropriate control sections to compare and ensure reproducibility of staining patterns.

Optimizing Antibody Concentrations:Titrate both primary and secondary antibodies to find the optimal concentrations that provide specific staining without background.

Proper Reagent Handling:Always use fresh and properly stored reagents to ensure consistent results. Avoid repeated freeze-thaw cycles of antibodies and other reagents.

Troubleshooting

If uneven staining persists, consider the following additional troubleshooting steps:

Check Antibody Penetration: For thicker sections, ensure that antibodies can penetrate evenly. You might need to increase the incubation time or gently agitate the sections during incubation.
Consistency in Section Thickness: Ensure all sections are of uniform thickness, as variability can affect antibody penetration and staining intensity.
Reduce Section Folding: Carefully monitor sections during handling to prevent folding, which can result in uneven staining.

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