My primary antibody is bet- galactosidase (biotin-conjugate)
I am not getting primary Ab binding but all my slides are stained dark with secondary and non specific binding. Should I use mouse on mouse kit? if yes, then which one?
Or should I do something else?
Please help
You are using a biotin-conjugated primary but are using an anti-mouse secondary. With a biotin conjugated primary there is no need to use an anti-mouse secondary as this step is there to add biotin to your primary, which you already have. You should add your primary antibody, then go straight to the avidin/biotin enzyme complex step, then to the substrate step.
By bathing your tissue in the anti-mouse secondary, you are introducing biotin to the tissue and then your next step then labels all that extra biotin, resulting in your non-specific stain. You may also want to consider antigen retireval. Is you r tissue fixed or frozen?
Renee is right, after biotinylated primary, directly go to ABC kit and DAB steps. (In your case, secondary is not doing anything and is unnecessary step). Be sure you are using the right ABC kit, verify the kit number with technical advisor of the company just in case of doubt. If you are using paraffin-sections, antigen retrieval is important.
All of the above is right to the point,
But antigen retrieval it´s a must.
I am using a formalin fixed ovary section
I tried with Tris/EDTA and Sodium Citrate both but had non specific results and high background
therefore I now tried with no antigen retriveal method
How will I know if my primary is working and that I dont have to change it???
Primary not good, that is just one possibility (contact other researcher who has used this antibody if that antibody worked for him/her), but there could be other factors too:
Check the antibody instructions, which antigen retrieval method is recommended,
Second, dilution of primary
Third, blocking buffer,
Last, increase washings after primary to get rid of binding from non-specific area.
If i am not using secondary antibody, then i also dont need to block the non-specific secondary in NGS as well I assume...
I am using 5% NGS to block non specific secondary
but since it’s established that I don’t need a secondary then what should I do in this case?
Originally I was using NGS as a block for secondary
i was using blocking solution for my negative control and primary antibody in a block as dilution for my positive control
now since I don’t have secondary, should I skip this step? And if I am skipping the block what should I use my control as ?? Just in PBS?
Hi Muniza,
Pl see some reading material on this website, that will clarify some doubts. This site is very useful, otherwise:
http://www.ihcworld.com/_technical_tips/biotin_tips.htm
Best regards,
Thank you.
My tissue does not contain biotin but my primary antibody does.
In this situation I am not going to use anti mouse secondary antibody cause it also has biotin.
If I am skipping the blocking step for secondary antibody what should I use as my control instead? Also, when you dilute primary with blocking solution what should I use Instead of that blocking solution?
Muniza,
Most of these ABC kits include 2.5% normal horse serum. You should use this as your diluent/blocker. Basic protocol:
Deparaffinize and Rehydrate
Antigen Retrieval
Block endogenous activity (either AP or HRP, depending on your kit Vector includes BLOXALL which blocks both AP and HRP).
Protein Block (use 2.5 NHS or skip this step)
Primary incubation (in 2.5% Normal horse serum provided in kit) overnight at 4 degrees C or 1 hr at 37 degrees C
Wash in buffer
Incubate 30 min in ABC Reagent provided in kit
Wash
Incubate in substrate solution (incubation dependent on substrate and detection system used)
Rinse in TAP water
Counterstain (incubation dependent on counterstain selected)
Clear and Mount
Renee
Hi
i performed exactly same experiment
using DAB but no staining :(
i am now trying with the secondary antibody but different concentrations just to check whether my primary has not that sufficient amount of biotin
i am very depressed
Muniza,
Did you try using antigen retrieval? This may be necessary for your antigen.
Renee
I did antigen retrieval as well
maybe I think I need to incubate it more time in the ABC
With high (Tris/EDTA) pH and low (sodium citrate) pH
I am using B-galactosidase monoclonal biotin conjugate antibody (sigma B-0271, 0.2ml,Lot# 096M4764)
Today I am going to try incubating my sections with 2 hrs incubation of primary and 4 hrs incubation with ABC (vectastain)
also, i am going to increase the conc of vectastain
Hi Muniza,
First, the sections you are using are frozen sections or formalin or PFA fixed?
If you are using frozen sections then you don’t need any antigen retrieval however if the sections are fixed ones, then make sure that these are not over fixed in 4% PFA or 10% neutral buffered formalin for more than 48hrs. A prolonged fixation in PFA or formalin will result in failure of antigen retrieval and hence no staining. Antigen retrieval is the most crucial step of IHC
Secondly, If your protein of interest is cell surface protein then retrieve your antigen by antigen retrieval by boiling your slide in sodium citrate buffer pH: 6 OR Tri-sodium citrate/EDTA pH:8 for 5min in microwave oven at high fire.
However if you protein is a nuclear membrane protein then above heat mediated antigen retrieval will not work. In this case 20ug/ml of proteinase-K is used for 7-10min at RT.
For Negative control, you can skip incubation of primary antibody and directly incubate secondary Ab and this will let you know whether the background resulted was because of non-specific binding of secondary antibody
Normally Primary antibodies are incubated O/N at 4 deg C and secondary Ab for 1hrs are RT,
Hope it helps
How long were they fixed in formalin Muniza? As Nasir Abbas pointed out above, the duration of fixation is extremely important.
They are fixed in formalin for 6 hrs. I am using citrate as a low pH antigen retrieval method. My gene is ubiquitously present in my tissue
Can you show us the staining you are seeing as well as your negative control staining?
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