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Speed Staining!

With the Olympics just past, questions such as how high and how fast come to the fore. So here’s our little IHC mini Olympics.

 

How High… can you dilute out your primary antibody?  

Primary antibodies are usually the most expensive part of an IHC stain, they determine the specificity of the test and if they are not well optimised with the staining system your staining may well fall short of the bar. Apart from temperature & incubation time, the dilution achieved is normally dependent on the detection method and also on the sensitivity of the substrate used.

 

How Fast

Generally the fewer steps in a method the faster it is. Although there are recommended staining methods with most kits, some can come with modifications of increased concentrations and higher incubation temperatures that allow for a faster method. Medal rankings based on standard method.

 

How controlled?

Sometimes a balance needs to be found between strength and precision.  As with sports, you also have to deliver in the test, as well as in ‘training’.

Although more layers can increase sensitivity, each can introduce more opportunity for unwanted background and then it takes more to With each layer, any existing unwanted reactions are also amplified, and usually cost!

 

Detection Method Medal Table

 

Event

Detection Methods

Highest Primary Dilution (Sensitivity)

Fastest Method

Ease of trouble shooting / background control

Examples

Polymer/ ABC & Tyramide

1

4

3

Biotinyl Tyramide [Signal Amplifier], ( tyramide) *

Amplified Polymer

2

3

2

ImmPRESS Excel

Polymer

3

1

1

HistoFine

ImmPRESS

ZytoChem Polymer

 

ABC (with Secondary)

3

2**

2

VECTASTAIN

Rockland ABC

Zytochem

Labelled Primary & HRP Streptavidin

4

1

1

Biotinylated Primaries

HRP Streptavidin

** A rapid staining method is available

Photo finish

 

At the end of the method finishing the sections for imaging is also important and can impact on the time taken, although there are restrictions as to appropriate mountants based on chemical compatibility which must be the primary consideration.

 

 

Event

Section Mounting

Speed to microscope post mounting

Longevity

Examples

Permanent (Non-Aqueous)

Dehydration Required

3

1

 

VectaMount

 

Non-Aqueous Express

1

2

VectaMount Express

Aqueous

1

3

Glycerol

VectaMount AQ

 

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