Dear Mai Tran,

You need to be really lucky to do successful immunofluorescence on these samples. Below I can give you a protocol that you can try. However, the chances for success will be rather small.

The fixation steps that have been performed will have destroyed most of the epitopes (an epitope is the 3D-protein sequence recognized by the antibody). Had you only used formaldehyde for fixation, it might have been possible. Glutaraldehyde destroys man more epitopes and OsO4 does the utmost damage. Again, I know only one or two antibodies that are able to detect such a fixed protein. Most of the Ab I have used does not even bind to formalin fixed proteins.

Standard protocol. If I understand correctly you consider staining for light microscopy.

(ultra)cut the specimen. I hope it is not in EPON or another hydrophobic resin.

100nm is a good thickness.

Lift the section w/ a loop from the ultra-pure water in the knives trough.

Have another drop of water on top of a poly-L-lysine coated glass slide.

Let the drop w/ the floating section dry slowly. Max 37°C

Draw a pap-ring around the section

Block for a-specific background by 1h to overnight incubation 1% ovalbumine or BSA in PBS. That means, simply put a drop of this block buffer on the section.

Dilute the 1st Ab in block buffer and apply it to the section Usually 50-100 ul is sufficient. Incubate 1h at room temperature. Eventually pray for good results J

Wash several times w/ PBS

Apply the secondary Ab as above

Wash

Nuclear staining w/ Hoechst is possible and will make it easier to find the section back under the microscope.

Wash

Watch

If this did not work you might consider putting extra time in this specimen (etching and/or antigen retrieval). But you rather start from zero with differently fixed material.

Kind regard,

Duco Kramer

Dear Mai Tran,

You need to be really lucky to do successful immunofluorescence on these samples. Below I can give you a protocol that you can try. However, the chances for success will be rather small.

The fixation steps that have been performed will have destroyed most of the epitopes (an epitope is the 3D-protein sequence recognized by the antibody). Had you only used formaldehyde for fixation, it might have been possible. Glutaraldehyde destroys man more epitopes and OsO4 does the utmost damage. Again, I know only one or two antibodies that are able to detect such a fixed protein. Most of the Ab I have used does not even bind to formalin fixed proteins.

Standard protocol. If I understand correctly you consider staining for light microscopy.

(ultra)cut the specimen. I hope it is not in EPON or another hydrophobic resin.

100nm is a good thickness.

Lift the section w/ a loop from the ultra-pure water in the knives trough.

Have another drop of water on top of a poly-L-lysine coated glass slide.

Let the drop w/ the floating section dry slowly. Max 37°C

Draw a pap-ring around the section

Block for a-specific background by 1h to overnight incubation 1% ovalbumine or BSA in PBS. That means, simply put a drop of this block buffer on the section.

Dilute the 1st Ab in block buffer and apply it to the section Usually 50-100 ul is sufficient. Incubate 1h at room temperature. Eventually pray for good results J

Wash several times w/ PBS

Apply the secondary Ab as above

Wash

Nuclear staining w/ Hoechst is possible and will make it easier to find the section back under the microscope.

Wash

Watch

If this did not work you might consider putting extra time in this specimen (etching and/or antigen retrieval). But you rather start from zero with differently fixed material.

Kind regard,

Duco Kramer

Dear Mai Tran,

this is a difficult task on these samples as already mentioned by Duco Kramer - as it is well known and accepted in the "Immuno" localisation field - especially EM - that Glutaraldehyde usually damages the "Antigen Conformation" in such a way that antibodies (which are usually selected for PFA fixed material) can not bind anymore.  OsO4 is even more damaging antigens epitope in such a way that no antibodies can bind except OsO4 has been used during a freeze-substitution and removed at low temperature.

If you need to stick to your EM preparation protocols - you need to lower the Glutaraldehyde concentration below 0,05% , avoid OsO4 and then most antibodies, working under histological conditions, may work. But you need to accept to test them with different concentration etc.. (a lot of work) - more details on this topic you can also find in the book from Gareth Griffith - "Fine Structure Immunocytochmistry" - Springer 1993 - it is still one of the best for ImmunoEM introductions and contains several working protocols eg. for Tokuyashu methods and some exploration to high end methods based on Cryo-Fixation.

If possible get some fresh tissue and prepare it for histology. If not possible use selected antibodies from groups, which successful worked on EM-fixed samples (ideally without GA and OsO4).

Otherwise you will loose a lot of time and it is not clear that you get any results - Immunolocalisation on "EM samples" is a hard business and needs a lot of time and experience for testing antibodies.

Histological staining (HE, Toluoidin  blue etc) still work on EM prepared embedded (also Epon resin)  - you need to etch them short with H2O2  (30%) for a few minute and then apply your colour staining solution at least over night - you will need a good colour digital camera on an light microscope to image such stained sections (we usually use 100-200nm ultra thin section) - but the resolution you get is astonishing since it is better then confocal imaging (the section thickness is better then the axial resolution of a CLSM). It may be that some antibodies will bind after the H2O2 treatment but as mentioned above it is very time consuming to test this.

with best regards

Roger

You may not be able to work on the samples you have. Subsequently, if you have deep freeze facilities available, you may on collecting fresh samples, divide them in three parts; one for EM, another for routine processing and the last in freeze storage. 

Unfortunately, no one processing method is ideal for the different techniques utilized by histologists/histotechnicians or in the field of cell or tissue biology. The solution lies in preserving the sample to be studied in frozen media.

Good luck

Dear Mai Tran,

I agree with the three answers that you have already received (above), in that you will probably spend much more time trying to get a protocol to work in a way that you trust on the material you already have, particularly since it has already been osmicated.  The fixation used for immuno-EM needs to be planned from the very beginning of the experiment to get good results.  Even then, it can be a challenge to get good labeling, even with immunogold techniques. 

Having said that, it may be that you have very rare specimens, and can't obtain new materials.   There was a time in the 1980's that investigators were trying to apply immunohistochemical techniques to materials prepared for EM.  In addition to the book recommended by Roger,  there are some methods that are suggested for archival material in this older review article by John Stirling (attached).  They are in the Etching and Antigen Unmasking section, but I add the caveat that they are to be applied to material already embedded in epoxy resin.

Good Luck!

Jill

Thank you for responding. I really appreciate the feedback. I will reconsider whether or not it is that critical to try immunostaining on these sections.

First of all you have to get any tissue specific antibody tagged with an immunofluorescent  agent, then stain the tissue section for your EM.You may follow any published report for this as well.

Dear Mai Tran,

I totally agree with the previous advices but You can find some exceptions in literature. Generally, epoxide polymers can be used in protein and sugar antigens.

Let's have some examples:

1. http://www.imaging-git.com/science/electron-and-ion-microscopy/making-new-out-gold (in this case high pressure freezing followed by freeze substitution is prerequisite)

but conventional TEM procedures is also useful:

2. detection of polysaccharides: Immunocytochemical location of the (1→3) (1→4)-β-glucan lichenin in the lichen-forming ascomycete Cetraria islandica (Icelandic moss) (DOI: 10.1046/j.1469-8137.2001.00122.x)

3. detection of proteins: PaCS Is a Novel Cytoplasmic Structure Containing Functional Proteasome and Inducible by Cytokines/Trophic Factors (doi:10.1371/journal.pone.0082560)

What is interesting, You can find within this article section entitled "Aldehyde–osmium fixation is required for PaCS detection by TEM or confocal microscopy". Thus, detection of a chosen antigen is dependent of its nature.

These article concern immunogold labelling but I think fluorescence detection also should works. Am I wright?

All the best,

Lukasz Mielanczyk

Dear Mai Tran,

We regularly etched and immunostained semithin sections, however not all antibodies worked because of the severe fixation mentioned above and only PAP or ABC techniques were suitable ie not immunifluorescence. The protocol is not difficult and may be worth a try.

Firstly prepare a saturated solution of NaOH in EtOH, this takes a while to 'ripen' indicated when it turns amber.

semithin 1um sections were etched in 50%solution above in EtOH 30mins with agitation (check under microscope for removal of resin)

rinse in EtOH several times and wash in running water 30mins

proceed with PAP or ABC technique.

Following the DAB reaction the product was enhanced by OsO4 for 1min.

Hope  this helps

Jennifer Barrie