[Histonet] IF staining questions (Charles Riley)

[Carl Hobbs]

The primary antibody is the most expensive reagent
If you use the primary ab unconjugated and visualise using a conjugated secondary you will significantly increase the dilution factor of the primary ab thus your primary ab will last much longer ( saving you money......and, maybe less background noise
Same principle applies to using antigen retrieval( AR) v no ag AR:
eg: I can use  a particular ab to detect "endogenous" GFP ( actually tagged to a protein) in Pwax sections without using AR ( actually HIER) at a dilution factor of 1/750
If I subject the section to HIER, I can use the same primary ab at a dilution factor of at least 1/2K
Same applies to 90C HIER v M/W HIER: former requires 30 mins or more, latter requires ½ that time and, is more effective overall ( stronger signal/greater diln factor)
Imho
Good luck!

Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson SPaRC
Guys Campus, London Bridge
Kings College London
London
SE1 1UL

020 7848 6810