Dear Harsh Nitin Dongre

(& well respected posting colleagues),

SHORT Version of my reply (only keeping in mind the issue of "visibility" of the embedded tissue block): use PPD (para-phenylene-diamine) in dehydration of your specs ( eg. 1% PPD in EtOH 70%, followed by washing at least twice in pure EtOH70%) [More about: see below]

yYour question unfortunately lacks some information on the purpose of your TEM-preparation which would be helpful for any colleague replying to it (tissue?, general processing-practical or intended?, ImmGold-Labeling pre-/ postembedding?, cryomethods?, understanding of function and reaction mechanism of OsO4 in tissue preps, etc., guessing you are using DURCUPAN-ARALDITE-resin embedding, right?).

[OK, now that I have written the post at home, I found your reply "I tried 3% GA....", so I have added also the short version above!) .

But we all would love to read about the (hopefully positive) outcome of your ImmGoldlabelling after 3% Ga-fixation!

As usual, the procedure(s) you can use will depend on what your goal in the end will be.

Fixation of specimens for (T)EM and immunogoldlabelling studies later on usually follows standard procedures which easily can be found either in relevant books on Techniques and Methods in EM, via PubMed or via Googleing.

“*Plain, buffered formaldehyde fixative*”, perhaps with a really small amount/concentration of glutardialdehyde (like the PLP= periodate – lysine- paraformaldehyde fixative[J Histochem Cytochem December 1974 vol. 22 no. 12 1077-1083 by McLean& Nakane] as mentioned above by Elizabeth) will result – *if done properly* – in really good ultrastructural preservation – not only in terms of antigenicity but also in terms of structures (important: no rapid dehydration like 70% - 90/96% - 100% EtOH!). Since for postembedding imm-labelling osmium usually is omitted, the ultrastructural will not be the expected one as after full classical TEM processing, especially one can expect only faint contrast after the standard staining methods, which is evident when observing sections in the TEM..

The use of Osmium tetroxide for postembedding ICC-labeling often is not recommended (epitope alteration/destruction, inconsistent for correct polymerisation of some resins like Lowicryls, but not for epoxides like araldite or Epon and its substitutes, usually polymerized by extrinsic heat treatment= polymerisation oven) but “oxidation” and “removal of OsO4(= oxidation of realisticly an amount of Os = OsO2 = Osmium black bound to proteins and fat) ” from ultrathin sections prior to immunolabeling (e.g. by periodic acid treatment) has been a possibility for many years in earlier times. It (using or omitting OsO4) also will depend on the antigens you try to (immuno-)localize.

Os-binding in your tissue material is not a “particle-matter” on observation in TEM competing with eventually bound gold particles from immunolabelling, but rather an issue of inelastic and elastic scattering of electrons under the e-beam. And: remember: when using 1% OsO4-solution for osmication, only (I hope to remember this quote correctly) 0.025% of atomic Osmium will be left bound to/in your tissue, if after osmication your samples have been washed with the appropriate buffer prior to first step of dehydration.

Since there have been some earlier question-threads in ResearchGate regarding this matter, I would like to point you there for reading (and perhaps get answers too):

https://www.researchgate.net/post/Chemical_fixation_for_TEM_samples-Os_alternative

https://www.researchgate.net/post/Does_anyone_have_a_sucessful_immunogold_pre-embedding_labeling_procedure_for_transmission_electron_microscopy_I

https://www.researchgate.net/post/Cell_monolayer_Electron_Microscope_protocol?

https://www.researchgate.net/post/how_to_prepare_sample_for_TEM?ev=home_person_post_question_object

https://www.researchgate.net/post/Is_there_a_protocol_for_Cacodylate_and_Glutaraldehyde_as_fixatives_for_cultured_cells

At:

http://cellbio.med.harvard.edu/research/research-facilities/conventional-electron-microscopy

you will find an interesting information, which will solve your problem with “invisibility of specimens after embedding into resin”:

Osmium Free Method for Resin embedding (Tannic Acid / Uranyl Acetate method)

1. Fix in PFA/ Picric acid as above. (NB: this fixative you’ll find on the webpage too)

Do the following processing on ice until 100% Ethanol:

2. Freshly made 1% Tannic Acid in Maleate Buffer (MB) for 40 min, then rinse 2x in MB

3. In the dark: 1% Uranyl Acetate in MB for 40 min, then rinse twice in MB

4. Dehydrate 5 min each: 50%/70% Ethanol

5. 1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min *)

6. Rinse twice in 70% Ethanol

7. Dehydrate 5 min each: 80%/95%/2x100% Ethanol

8. Resin infiltration (LR white or other hydrophilic resin)

9. Polymerization at 40° C

*) p-phenylenediamine will your tissue (no problems in terms of retained antigenicity) with a brown to dark-brownish-black coloration.

General Reading: Tissue processing by: Leigh Winsor

( http://www.scientistsolutions.com/data/modules/protocols/images/0795c499-85a4-4018-afb1-433fdf615395/8aec7959-df18-4b2e-949b-fc62124e2ef7/tissue processing.doc )

If we knew a little bit more about your tissue, processing and your intended Imm-labeling most of us could be more of help, I guess.

Best wishes for the weekend and the progress of your work,

regards, Wolfgang

Did you try a mixture of glutardialdehyd/formaldehyde?

GLUTARALDEHYDE/PARAFORMALDEHYDE MIXTURES

Lower concentrations of GA (0.02 – 0.2%) mixed with 4% formaldehyde are preferred when preparing samples for immunolocalization.

An alternative is periodate paraformaldehyde (J Histochem Cytochem December 1974 vol. 22 no. 12 1077-1083) by McLean& Nakane

with respect to the visibility.

Did you think of post-vital staining after aldehyde fixation?

If you do pre-embedding immunogold labeling, the OsO4 does not interfere with your gold labeled proteins.

It is true that OsO4 puts the tissue black, but this is only when you look it with your eyes or with an optical microscope. If you put that tissue into the transmission electron microscope you could check that your sample is not black.

Your gold labeled proteins will be black, just like you said before but you will be able to differentiate them from the rest of the tissue.

My recommendation is to use a fixation based in glutaraldehyd or formaldehyde. After that you can obtain vibratome sections, then do the immunogold labeling. Now is time for doing the OsO4 fixation and finally make the embedding into the resin.

I tried 3 % glutaraldehyde both before immunogold labeling and after it. Awaiting for the results of it.

I dont have problem with fixation. Only problem is I cant differentiate between the tissue(not OsO4 treated) and the araldite block. And hence it is very difficult to perform proper block trimming and obtaining ultrathin sections.

I will perform pre-embedding method as suggested by you and let you know about the results.

Use 2% uranyl acetate as a post-fixative and embed in an acrylic resin such as LR White. You may wish to reduce the glutaraldehyde concentration to 1% as well.

While trimming into the block, use a mirror, mounted on the knife holder and tilted at about 45 degrees, to see light reflected off the block surface; you will be able to see the sample easily due to roughing down artefact. Cut a 0.35 - 0.5 um section and stain with toluidine blue to confirm your area of interest before cutting thin sections.

You can try 1% Tannic Acid (in water) followed by 1% Uranyl Acetate in place of the osmium.

Dear Harsh Nitin Dongre

(& well respected posting colleagues),

SHORT Version of my reply (only keeping in mind the issue of "visibility" of the embedded tissue block): use PPD (para-phenylene-diamine) in dehydration of your specs ( eg. 1% PPD in EtOH 70%, followed by washing at least twice in pure EtOH70%) [More about: see below]

yYour question unfortunately lacks some information on the purpose of your TEM-preparation which would be helpful for any colleague replying to it (tissue?, general processing-practical or intended?, ImmGold-Labeling pre-/ postembedding?, cryomethods?, understanding of function and reaction mechanism of OsO4 in tissue preps, etc., guessing you are using DURCUPAN-ARALDITE-resin embedding, right?).

[OK, now that I have written the post at home, I found your reply "I tried 3% GA....", so I have added also the short version above!) .

But we all would love to read about the (hopefully positive) outcome of your ImmGoldlabelling after 3% Ga-fixation!

As usual, the procedure(s) you can use will depend on what your goal in the end will be.

Fixation of specimens for (T)EM and immunogoldlabelling studies later on usually follows standard procedures which easily can be found either in relevant books on Techniques and Methods in EM, via PubMed or via Googleing.

“*Plain, buffered formaldehyde fixative*”, perhaps with a really small amount/concentration of glutardialdehyde (like the PLP= periodate – lysine- paraformaldehyde fixative[J Histochem Cytochem December 1974 vol. 22 no. 12 1077-1083 by McLean& Nakane] as mentioned above by Elizabeth) will result – *if done properly* – in really good ultrastructural preservation – not only in terms of antigenicity but also in terms of structures (important: no rapid dehydration like 70% - 90/96% - 100% EtOH!). Since for postembedding imm-labelling osmium usually is omitted, the ultrastructural will not be the expected one as after full classical TEM processing, especially one can expect only faint contrast after the standard staining methods, which is evident when observing sections in the TEM..

The use of Osmium tetroxide for postembedding ICC-labeling often is not recommended (epitope alteration/destruction, inconsistent for correct polymerisation of some resins like Lowicryls, but not for epoxides like araldite or Epon and its substitutes, usually polymerized by extrinsic heat treatment= polymerisation oven) but “oxidation” and “removal of OsO4(= oxidation of realisticly an amount of Os = OsO2 = Osmium black bound to proteins and fat) ” from ultrathin sections prior to immunolabeling (e.g. by periodic acid treatment) has been a possibility for many years in earlier times. It (using or omitting OsO4) also will depend on the antigens you try to (immuno-)localize.

Os-binding in your tissue material is not a “particle-matter” on observation in TEM competing with eventually bound gold particles from immunolabelling, but rather an issue of inelastic and elastic scattering of electrons under the e-beam. And: remember: when using 1% OsO4-solution for osmication, only (I hope to remember this quote correctly) 0.025% of atomic Osmium will be left bound to/in your tissue, if after osmication your samples have been washed with the appropriate buffer prior to first step of dehydration.

Since there have been some earlier question-threads in ResearchGate regarding this matter, I would like to point you there for reading (and perhaps get answers too):

https://www.researchgate.net/post/Chemical_fixation_for_TEM_samples-Os_alternative

https://www.researchgate.net/post/Does_anyone_have_a_sucessful_immunogold_pre-embedding_labeling_procedure_for_transmission_electron_microscopy_I

https://www.researchgate.net/post/Cell_monolayer_Electron_Microscope_protocol?

https://www.researchgate.net/post/how_to_prepare_sample_for_TEM?ev=home_person_post_question_object

https://www.researchgate.net/post/Is_there_a_protocol_for_Cacodylate_and_Glutaraldehyde_as_fixatives_for_cultured_cells

At:

http://cellbio.med.harvard.edu/research/research-facilities/conventional-electron-microscopy

you will find an interesting information, which will solve your problem with “invisibility of specimens after embedding into resin”:

Osmium Free Method for Resin embedding (Tannic Acid / Uranyl Acetate method)

1. Fix in PFA/ Picric acid as above. (NB: this fixative you’ll find on the webpage too)

Do the following processing on ice until 100% Ethanol:

2. Freshly made 1% Tannic Acid in Maleate Buffer (MB) for 40 min, then rinse 2x in MB

3. In the dark: 1% Uranyl Acetate in MB for 40 min, then rinse twice in MB

4. Dehydrate 5 min each: 50%/70% Ethanol

5. 1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min *)

6. Rinse twice in 70% Ethanol

7. Dehydrate 5 min each: 80%/95%/2x100% Ethanol

8. Resin infiltration (LR white or other hydrophilic resin)

9. Polymerization at 40° C

*) p-phenylenediamine will your tissue (no problems in terms of retained antigenicity) with a brown to dark-brownish-black coloration.

General Reading: Tissue processing by: Leigh Winsor

( http://www.scientistsolutions.com/data/modules/protocols/images/0795c499-85a4-4018-afb1-433fdf615395/8aec7959-df18-4b2e-949b-fc62124e2ef7/tissue processing.doc )

If we knew a little bit more about your tissue, processing and your intended Imm-labeling most of us could be more of help, I guess.

Best wishes for the weekend and the progress of your work,

regards, Wolfgang

When I would have choice I would stay as far as possible from OsO4.

With a lot of luck you will retain stable epitopes. For conformational ones the most mild method in water miscible crosslinking resins is best.

There is a good review in J. Immunological methods "State of the art in antigen retrieval for immunohistochemistry."J Immunol Methods. 2009 Feb 28;341(1-2):1-18. doi: 10.1016/j.jim.2008.11.007. Epub 2008 Dec 6.

Harsh,

If all you want to do is differentiate between tissue and the araldite block then try using tannic acid as a secondary fixative (0.13%-0.25% for IEM) followed by 2% uranyl acetate. This should render the tissue brown. I would be very careful about 3% GA for IEM, you are probably going to over cross-link the epitopes, and if not quenched with glycine or ammonia chloride then you could have some aldehyde groups hanging around which could cross-link your Aby (false positive). You can try reduced Osmium after a light PF GA fix, and then do some etching on the grid with sodium m periodate to remove the osmium and make epitopes accessible (works with really hardy epitopes).

good luck,

Michael Delannoy