Asynchronous solid phase ELISA sandwich was developed:

The 96-year-old black-water polystyrene plates were painted overnight at 4 ° C with monoclonal antibodies AT 180 and AT 270 in 50 million NaHCO3, pH 9.6. The mAb AT 180 and AT 270 directed against the rings containing the phosphoresities Thr-231 and Thr-181, respectively, are weakly interacting with the natural taw. After washing 3 times with PBS containing 0.05% Tween, 100 μl of brain homogenization per well was incubated for 3 hours at room temperature. After washing 3 times with PBS-T, mAb AD2-labeled mAb was added in PBS-T for two hours at room temperature, followed by 3 other wash steps with PBS-T. The reporter mAb AD2 does not show any interaction with the normal brain of the old brain and discovers phosphorylated Ser-396 and Ser404. One hundred μl of the chemiluminescent substrate was added to the wells and the relative lighting units were read after 5 minutes on the ELISA Luminometer. Each plate comprises a standard dilution series of thosphorilated recombinant human tau in a homogenization buffer. The recombinant tau protein was recombinant with the brain homogenis.

Hadeel Kamil ?????

Dear Abhishek.

There are some steps missing in your post - I do not know wether you forgot to mention or if you did not perform these steps.

Some background information regarding DAB staining:

https://www.abcam.com/kits/dab-staining

https://www.bosterbio.com/protocol-and-troubleshooting/immunohistochemistry-ihc-principle

You could try this:

- Wash your slides in PBS thoroughly (3x5min)

- You have to incubate your slides in H2O2 in order to deactivate endogenous peroxides (this step is essential for DAB-stainings, since the reaction is started by adding H2O2). 10 minutes 3%H2O2 in PBS should be sufficient.

- Wash your slides in PBS thoroughly (3x5min)

- Antigen Retrieval

- Wash your slides in PBS thoroughly (3x5min)

- Blocking: Also an essential step to prevent unspecific binding of your antibody. Depending on your secondary you can use f.e. normal horse serum for secondaries made in horse: 10% NHS in PBS, incubation time 1 hour at room temperature.

- Application of your primary in different concentrations (we dilute them in 3% NHS/PBS solution). Incubation time: over night at 4°C.

- Wash your slides in PBS thoroughly (3x5min)

- Application of your biotinylated secondary f.e. Horse anti-mouse IgG (for Vector secondaries we dilute them 1:150 in PBS). Incubation time: 1 hour at room temperature. You can also use HRP-conjugated secondaries, then the next step is negligible.

- ABC (Avidin Biotin Complex with peroxidase): this is essential for visualization of your antigen-antibody reaction if you use biotinylated secondaries! If you use the ABC-Kit from Vector you have to preincubate it 30 minutes before application. Incubation time: 1h at room temperature

Background: the biotin of your secondary forms a complex with the avidin in the kit which is conjugated with a peroxidase.

- Wash your slides in PBS thoroughly (3x5min)

- DAB (caution: cancerous!)

- Start the reaction by adding H2O2 -> the slides get brown. Check the intensity of the color under the microscope.

Background: The DAB is oxidized by H2O2 in a reaction catalized by peroxidase and forms a brown precipitate at the location of the peroxidase.

(Don´t forget to deactivate the DAB:

f.e. https://extranet.fredhutch.org/en/u/ehs/hamm/chap6/section3.html)

- Wash your slides in water thoroughly (change the water at least 5 times)

- Mount your slides on glass slides using gelatin

- Dry them for 15-30 minutes

- Dehydrate in ascending alcohol series.

- Dehydrate in xylene

- Coverslip f.e. with DPX.

For protocols (or concentrations of your primaries used by others) you could check the manufacturer´s webpage for more information to start with. In many cases they refer to publications in which the antibodies have been used.

F.e.: https://www.thermofisher.com/antibody/product/Phospho-Tau-Thr181-Antibody-clone-AT270-Monoclonal/MN1050

leeds to

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362695/figure/F3/

I do not know what animal model you use, but if you use mice, then there are some pitfalls if you use mouse-antibodies on mouse tissue...

My two cents regarding DAB-stainings: they are prone to unspecific background staining and can not be used for double- or triplelabeling (f.e. if you want to see in what cells the questionable antigen is located, by using a known cellular marker). If you have a fluorescence microscope in your facility I would go for immunofluorescent stainings. They are easier to perform and not so "mutagenic".

Hope this helps.

Marcia Gasis hi

Thankyou for the detail answer.

And yes i did try all the above mentioned method you listed (sorry i didnt put them all up ).

Yes i will be trying different antigen retrival method now. lets see.

anyways i am working on free floating sections.

KA models of sprague dawley rats.