Primary and secondary antibodies for immunostaining are supplied concentrated and must be diluted into a working solution before they touch tissue. Getting the volumes exactly right saves expensive reagent and keeps staining reproducible. This calculator turns a target dilution and a batch size into precise pipetting volumes.
How it works
A dilution written as 1:N means one part antibody brought up to N total parts. In a chosen working volume V the split is simply:
antibody volume = V / N
diluent volume = V - (V / N)The working volume itself is driven by how many sections you are staining plus an overage so you never run dry:
V_total = (sections x volume_per_section) + dead_volumeIf you would rather work from concentrations, the tool derives the dilution factor by putting both into the same units:
N = stock(ug/mL) / target(ug/mL)
= (stock_mg/mL x 1000) / target_ug/mLWorked example
To stain 10 sections at 100 microlitres each with a 50 microlitre overage, the total working volume is 1,050 microlitres. At 1:200 that is 5.25 microlitres of antibody made up with 1,044.75 microlitres of diluent. Reducing the antibody concentration raises that diluent portion; switching to 1:400 for comparison means just 2.625 microlitres of antibody in the same 1,050 microlitres.
Titration and lot-to-lot variation
Manufacturers recommend a starting dilution range, but the optimal point varies by tissue type, fixation protocol, antigen retrieval method and antibody lot. Standard practice is to run a small concentration series — for example 1:50, 1:100, 1:200 and 1:400 — on a control tissue that contains both positive and negative cells. Enter each dilution here to get exact pipetting volumes, then stain in parallel and compare signal-to-background ratio on a light microscope.
When a new lot arrives from the same supplier, re-titrate rather than assuming the same dilution holds; lot-to-lot variability is common enough that skipping this step produces inconsistent results across batches.
Dead volume and why it matters
Pipetting loss, evaporation from uncovered slides, and the need to fully cover each section without overflow mean you always prepare a little more working solution than the bare arithmetic requires. Adding 50–100 microlitres of dead volume is typical for most runs. For very high-value antibodies you might reduce it; for large automated stainer carousels the manufacturer often specifies a fixed dead volume per tray position.
Primary versus secondary antibody dilutions
The same arithmetic applies to both. Secondary antibodies are typically diluted in a broader range — commonly 1:200 to 1:1,000 — because they are less expensive and background accumulates if the concentration is too high. You can use this tool separately for each antibody used in a protocol to produce a full reagent-preparation sheet before starting a run.