Would you consider embedding the neuroshere? You may section it and then stain the slides to get the signals from the inside.

Try Triton-X100 before and during antibody incubations (During blocking step and Primary antibody step). So that it has much time to act and it does not interfere with antibody binding.

Thanks Dear Tamara..

Thanks for your answer Katie, but I thing it will be the toughest job to do, to cut sections of a neurosphere by a microtome after embedding in paraffin....You will never get a clue as which part of paraffin contans what.... They are microscopic as well as rare to isolate with perfect purity.....

Thanks Goodwin... I know triton is recommended with in the form of TBS during all washing and incubation steps for some kit based IS assays.. But triton and tween-20 are my standardized concentration during neural differentiation and they work really excellent with cells while unable to permeabilize the neurosphere..But your suggestion seems very logical and practical. Will try the same for sure..Thanks !

you may seed the whole sphere on cover slip which has been coated. i.e gelatin, pdl, plo. after over night, it will attached the coverslip as sphere. then fix,block and add antibody, I usually do it by this way.

Thanks dear Monica

What percent Triton-X and Tween are you using in your TBS? And do you use them in combination?

I am having the same problem, nestin looks great on the surface but any cytoplasmic markers do not penetrate the surface of the sphere. Should I up my concentration of Triton-X to 0.5% ? 

You may try NP40+TritonX100 with BSA in PBS for permeabilization step to overcome permeability issues...because they work on different set of proteins/lipids. Wash off these detergents and then use only TritonX100 containing PBS with BSA for immunostaining.

As you have seen, antibodies usually fail to penetrate fully into compact spheres with classical protocols of fixation and permeabilization. We developed a protocol allowing successful antibody penetration in tumor spheres of 150 μm in diameter and this allowed to perform whole mount immunostaining of spheres. This protocol is based on incubation with a solution containing PFA and Triton followed by dehydration/rehydration baths in methanol and long antibody incubation times: https://www.researchgate.net/publication/42388336_In_situ_protein_expression_in_tumour_spheres_Development_of_an_immunostaining_protocol_for_confocal_microscopy

You can also consider to prepare cryosections or paraffin sections but you need to concentrate the spheres in the mold if you want to easily localize your structures.

 Cryosections: The spheres are embedded in OCT. I usually place a cut tip on the center of the cryomold. I add the sphere solution in the tip and I discard the excess of liquid before adding OCT in the cut tip and around it. Then, I remove the tip so that the spheres are concentrated at the top of the OCT block, on the center. Finally, the cryomold is snap frozen in liquid nitrogen.

Paraffin sections: The spheres are fixed and washed three times with PBS. Then I let the spheres settle down on the bottom of 1.5 ml tube. I discard the excess of PBS before adding 50 μl of warm 2% agarose solution. I gently mix the solution and the spheroids using a micropipette with head-cut tips. After cooling, the gel containing the spheroids is unmolded and can be processed for paraffin embedding.

Article In situ protein expression in tumour spheres: Development of...

Thanks Louis..I followed a simpler method and I successfully published the paper...

Cheers !

Ajay Kumar Can you share me your paper in which you stained these spheres.

Hi Gurjeet. Please look at my molecular Neurobiology paper in my profile.