Diluents and rinse buffers for murine IHC - loonnnnng answer

From:Gayle Callis


We work strictly with frozen sections in our lab for all murine IHC/IFA
work and what we do may be different from others -  

For buffers, either PBS or TBS work. I do not use pure buffers to dilute
antibodies, but do use them for diluting Strepavidin-Alexa fluorochromes
from Molecular Probes. 

FOR IHC: All diluents/rinse buffers contain 0.05% Tween 20 and 0.2% normal
serum matched to host of secondary or for biotinylated Abs - we use goat or
swine or donkey serum. BSA can be used, we prefer serum.  We avoid rabbit,
sometimes use horse serum.
Our rule is dilute a biotinylated Rat antimouse in the normal serum block
(NSB) containing 10% goat/2.5% mouse/0.05% Tween 20.  You can vary mouse
serum conc from 1 to 5%. NSB is done BEFORE a primary Ab application, and
generally before the avidin/biotin block, although I know people who use it
after A/B block.

For a biotinylated hamster anti mouse ( make sure you know which hamster
species you primary is raised in either Armenian or Golden Syrian)- the NSB
is 10% goat, diluent is 1 - 2% goat without mouse serum. Hamster monoclonal
Abs seem to need no extra blocking with mouse serum. If a pure hamster
antimouse, the serum diluent is matched to host of secondary Ab.  

For unconjugated primary Ab i.e. Rat antiMouse, we use biotinylated goat
antiRat F(ab')2 secondary Ab adsorbed to mouse diluted in 10% goat serum
with 2.5% mouse and 0.05% Tween 20.  For Hamster antiMouse, we dilute
primary in 1% goat, and dilute the secondary donkey anti Hamster F(ab')2
frag of IgG, adsorbed to mouse in 10% goat.  We also select the Donkey anti
Hamster for species of Hamster the primary was raised in either Armenian or
Golden Syrian (Jackson Immunoresearch).  OR use Pharmingens Armenian/Golden
Syrian biotinylated cocktail as the secondary.  

In other words, the host of the secondary antibody determines the normal
serum block and what goes in the diluent.
Goat hosted secondary - goat serum
Donkey hosted secondary - donkey serum
In general, we stay away from rabbit secondary Abs. We tend to get more
background with sticky bunny Abs. If we did use them- we by adsorbed to
mouse and F(ab')2 frag of IgG. 

We seldom use BSA  or use Jacksons BSA, protease/immunoglobulin free or
high quality molecular biology grade - more often when staining a lectin.
Normal serums are heat inactivated and microfiltered for cleaner aggregate
free serum and sterile storage. 

We faithfully block when using either avidin or Strepavidin in the protocol
- no questions asked, it is a step we automatically use. We use either
Vector avidin/biotin kit or VECTOR strepavidin/biotin kit. Vector has a
tidy way to combine NSB with avidin and biotin in their package insert, it
save time, works great. 
We add avidin solution to normal serum block in such a way to not dilute
the normal serums. i.e. 2 ml avidin solution, 1 ml goat serum, 250 ul mouse
serum and qs to 10 ml to get a combination avidin/10% goat/2.5% mouse NSB,
apply for 15 minutes, rinse, go to biotin step. 
Do the same preparation for biotin block and incubate it for 15 min, cuts
down on time.  In general, avidin/biotin blocks (if worried about
endogenous biotin in serum, should be done AFTER the NSB to address endog
biotin in serum - but we never have much in the way of problems/background
- even on tissue with endog biotin). 

We never use commercial diluents usually because we don't know what is in
them.  Azide in a diluent for secondary antibody can inhibit HRP, beware! 

AS FOR IHC versus immunofluorescence - I tend to do less concentrated NSB
blocks 10% < to 5%, incubation time is the same. We frequently increase
primary Ab conc for IFA and confocal laser scanning microscopy.  Secondary
Ab concentrations may need to be increased - dependent on age of
fluorchrome - they get old! You need to determine all primary and secondary
concentrations by dilution panels.   

Example of our double IFA stain:  1) pure unconjugated primary
Ab/fluorochrome conj secondary Ab followed by 2) biotinylated primary Ab/SA
flurochrome - both Abs are rat anti mouse.  For immunofluorescent work we
never use O.05% Tween 20 unless called for and decrease NSB concentrations.
If we start to get background fluorescence, we will increase normal serum
block concentrations in NSB and diluent. 

Normal serum block 5% Donkey/1.25% mouse/Avidin 15 min
Rinse with Dulbeccos PBS/0.2% goat serum
Normal serum block 5% donkey/1.25% mouse/Biotin 15 min
Pure rat antimouse A diluted in 1% goat 30 min		
Rinse - "I realize I must avoid anything containing biotin, i.e. serum,
milk, BSA etc in order not to lower or block the binding."  We don't have
problems but worry more about background from binding to endog biotin.
Serum doesn't have a lot of biotin, I have poor success with casein/milk -
plus with the "white stuff" covering sections, I can't see them and get
very nervous - so I never use milk/casein, it is reserved for calves and
babies!  BSA works fine in buffers or diluent, but rarely use it and if I
do, still add normal serum matched to host of secondary i.e. a cocktail of
BSA/normal serum. 
Procedure for double IFA: 
Donkey antiRat F(ab')2 frag of IgG adsorbed to mouse conjugated to RRx i.e.
rhodamine (Jackson - brighter, more resistant to photobleaching) diluted in
5% donkey serum for 30 minutes
5% rat serum for 10 - 15 minutes (you must insure you don't get cross
reaction with the next rat antimouse primary. 
Biotinylated rat antimouse B diluted in 5% goat/1.25% mouse
Rinse with DPBS without serum or BSA to equilibrate section for next step 
Strepavidin-Alexa 488 (FITC equivalent, less photobleaching, brighter, very
clean) diluted in pure DPBS per Molecular Probes directions 
Rinse with DPBS x 5, coverslip with antifade mounting media. 

We tend to be purists, non kit users - working with each antibody
differently but that's research - we get clean, crisp results for IHC and
IFA.  All staining is done by hand, IFA staining cannot be done on
autostainers due to exposure to light. 

If you have any question, please contact me directly, I am off Histonet
starting today. 


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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