This page is dedicated for scientists to communicate, comment, share protocols and so on. If you would you like to share your protocol – please email it to us and we will post it below (with due credit, of course).

How to add your protocol –

We will need an email with the following details.

1. Full Name – upon request we can post initials only.
2. Email address – you can choose if to post it or not.
3. Full protocol – as you would publish in a scientific journal.
4. A result – a single image: Jpeg format, under 2MB.
5. Please be sure you have a PI and institute approval.

Invignome Team.

Invignome’s starter IHC protocol

Here is our simplified “start here” protocol – adapt it at will to your needs.

Preliminary steps

1. Before starting your protocol – allow the slides to reach room temperature in a closed slide box.
2. While the slides warm up prepare your buffers and reagents, dilute your antibodies to the right concentrations and so on.
3. Before starting – apply a hydrophibic barrier line around every slice:

• Shake the pen vigurously for 10 seconds, roughly.
• Remove the GnomePen cap off and make sure the tip is clean.
• Run a line around your samples, making sure to connect the circles or shapes you draw to generate a continuous hydrophobic barrier.
• Allow the line to dry off for a few seconds (usually under a minute).

Running the protocol

1. Apply your fixative by covering your sample with 2% Paraformaldehyde (or formalin, in a fume hood) and incubate for 20 minutes.
2. Wash your slices three times (usually rinse them three times – gently! – with PBS or TBS).
3. Wall perforation is needed to detect proteins that are internal expressed (i.e. not expressed or found on the cell wall externally) – to do so incubate your slices with PBS-T: PBS supplemented with 0.1% Triton X100, Triton X114 or Tween-20 for 20minutes.
4. Wash your slices three times
5. Apply your primary antibody, according to the manufacturer’s recommendation (usually dissolved in PBS or PBST for 30minutes to 2hours, for overnight incubations – store the slices at +4C)
6. Wash your slices three times.
7. Apply your Secondary (flourescent) antibody, according to the manufacturer’s recommendation (usually dissolved in PBS, TBS with or without a detergent – for 30minutes to 2hours, for overnight incubations – store the slices at +4C)
8. Wash your slices three times.
9. Apply a mounting media and cover your slices with a glass cover. The mounting media will harden and protect your slide.
10. Use a microscope with proper filters to examin your results.
11. Sore your slides at -20C for long term preservation.
12. Most importatntly – record your data!

Remember this is just a guideline – you will have to callibrate your own protocol to fit your antibodies, target proteins and so on. Don’t dispare and good luck with your experiment.

Thank you for visiting!

Invignome Team