Writing methods for immunocytochemistry or immunohistochemistry

As with every other subsection of your methods, you should include all the details about your experiment that are relevant to their interpretation. That’t everything, start to finish. As usual, use this list of points as a reference against which you can check your writing to make sure it’s complete.

Sample preparation

IHC
- Where the samples came from and how acquired (see ethics / patient recruitment section for human samples); age, disease/genotype etc
- Time from dissection to processing and/or post-mortem time to retrieval,
- How they were dissected, oriented etc
- Post acquisition processing – fixation, sucrose infiltration, dehydration procedures
- Embedding – paraffin. OCT etc
- Sectioning – thickness, microtome/cyrostat used, slide type
- Rehydration
- Antigen retrieval – time, chemical or enzyme, (pH if relevant), how (microwave, water bath etc)
ICC
- All the usual cell culture requirements (usually in their own section)
- Time from plating, density, and substrate; glass/plastic, thickness, coating etc
- Fixation; chemical, time, temperature
- Extraction; chemical, time, temperature
- Storage if relevant

Processing

Block; substance (goat serum), concentration, dilution, time and temperature of incubation
Primary antibody incubation; all the antibody details as usual (often in their own subsection), dilution and solution, incubation time, temperature.
Wash steps; solution, concentration, time
Secondary antibody incubations; all the antibody details, dilution, solution, incubation time and temperature,
Equivalent details for stains – concentrations, incubations, chemicals etc
Mounting a coverslipping – solutions, counterstains etc

Don’t forget to describe your controls – you should know what you need for your experiment, make sure to include full details here (and supplemental figures for review).
- non-specific bindings; pre immune serum, isotypes, antigen cleared)
- negative controls; different species, non-expressing cells- knockdown, knockout.
- positive controls; known cell lines/tissue with established distribution. Make sure to include the images if trying to prove a negative.

Imaging

Microscope information – manufacturer, objective, NA, immersion, illumination
Acquisition details – laser line details, filters, Z stack thickness, pinhole size, binning, scan direction, pixel, anything else relevant to analysis.
Camera details – manufacturer, colour profile, bit depth

Analysis

Don’t forget this bit!

How did you go from images to numbers? What did you quantify
If you didn’t quantify anything, how did you choose which images to use?
Which program did you use, including plug ins etc for FIJI (image J)
If any sort of intensity analysis; what did you use to calibrate; what were your internal controls? (fundamentally be careful here!)
If using an indirect or enzymatic approach, how did you account for non-stoichiometric binding
Which method of co-localisation quantification you used (Mander’s, Pearson’s etc) – you can’t say signals are co-distributed without the numbers to support it.
Usual statistical questions; number and types of replicates, technical, biological repeats
If you used pseudocolouring/LUTs describe the algorithm. (if you took images with a black and white camera, you should generally choose not to add unnecessary pseudocolour)