Problems with old samples and IHC

[Avi Jacob]

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Hey all,

I need some help troubleshooting an IHC protocol for a lab.
99% of what reaches my facility is fresh, or relatively fresh, from TC or
small animal experiments.

The samples we are having issues with are from *human brains* that have
been in *formalin for a number of years* (some for over a decade or two)
and, according to records, the tissues were not prepared immediately,
rather some were processed 48h post-mortem.

The IHC has seen little success: we are getting mostly background with
little specificity. A couple of samples do seem to have worked, but we
cannot rely on one or two slides (out of many dozens). In some samples,
even the dapi is not working well.
Here are some things that were tried by the lab in order to improve the IHC
protocol:

   - 24 h incubation in paraffin (as part of the paraffin block protocol).
   - Antigen retrieval with citraconic-anhydride acid (supposed to handle
   the formalin crossover).
   - Increased primary antibody conc. to 1:100.
   - TSA for secondary in order to strengthen the signal
   - Two different kinds of blocking were tried, both of which are supposed
   to work in this type of IHC:  CAS Block and  Goat serum
   - A *MaxBlock kit *is being used to lower the high autofluorescence
   inherent to these samples. *(Can this itself be blocking the a.b.'s? I
   never worked with this before and it gives a black tint to all the samples.
   Supposedly without this, the samples are impossible to image.)*


Does anyone have experience with such old samples and have successfully
done IHC with multiple colors?
Any tips or ideas are very much welcome.

Cheers,
Avi


--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit
Bar-Ilan University, Ramat-Gan 5290002, Israel

[Sylvie Le Guyader]

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Human, brain and postmortem... This is definitely a tough one.
I first would try and work at reducing the autofluorescence without staining the sample.
For example you can try bleaching the sample (e.g. exposing the sections overnight in a hood with a UV lamp). NAD, flavins... usually bleach very nicely. You can also try oxygen peroxide. Once you find a way to reduce autofluorescence, you optimize the staining.

I would guess that paraffine embedding would make things worse but if you need it, from what we have seen Soudan black seems to work well to reduce the fluorescence due to paraffine embedding. However it sounds like MaxBlock is similar.

You could try and use linear unmixing. Sometimes you cannot see your signal but if you manage to exclude the autofluorescence spectrum, you start finding out that your staining has worked a bit. Then it is easier to troubleshoot. You can acquire the 'pure' fluorophore spectrum by immunostaining cell in culture. Then you use this spectrum to unmix your real sample.

Good luck!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Avi Jacob
Sent: 19 October 2019 19:05
To: [hidden email]
Subject: Problems with old samples and IHC

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*****

Hey all,

I need some help troubleshooting an IHC protocol for a lab.
99% of what reaches my facility is fresh, or relatively fresh, from TC or small animal experiments.

The samples we are having issues with are from *human brains* that have been in *formalin for a number of years* (some for over a decade or two) and, according to records, the tissues were not prepared immediately, rather some were processed 48h post-mortem.

The IHC has seen little success: we are getting mostly background with little specificity. A couple of samples do seem to have worked, but we cannot rely on one or two slides (out of many dozens). In some samples, even the dapi is not working well.
Here are some things that were tried by the lab in order to improve the IHC
protocol:

   - 24 h incubation in paraffin (as part of the paraffin block protocol).
   - Antigen retrieval with citraconic-anhydride acid (supposed to handle
   the formalin crossover).
   - Increased primary antibody conc. to 1:100.
   - TSA for secondary in order to strengthen the signal
   - Two different kinds of blocking were tried, both of which are supposed
   to work in this type of IHC:  CAS Block and  Goat serum
   - A *MaxBlock kit *is being used to lower the high autofluorescence
   inherent to these samples. *(Can this itself be blocking the a.b.'s? I
   never worked with this before and it gives a black tint to all the samples.
   Supposedly without this, the samples are impossible to image.)*


Does anyone have experience with such old samples and have successfully done IHC with multiple colors?
Any tips or ideas are very much welcome.

Cheers,
Avi


--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit Bar-Ilan University, Ramat-Gan 5290002, Israel


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[Tim Feinstein]

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*****

With brain samples prepared under much more favorable conditions, I have had success spectrally unmixing the autouorescence using a 32-PMT Nikon A1 (no commercial interest). Leica and Zeiss scopes should have comparable functionality though Leica’s use a different mechanism.

Separately, if you can find a FLIM system then you may be able to isolate the antibody signal in the frequency domain, especially if you use probes with really distinct lifetimes. Lanthanides would be ideal but I’ve never seen those used for imaging, just plate reader FRET-FLIM.  Long-shift probes with built in FRET might also do. It’s a technical challenge but your particular needs may justify the trip and effort.

Best,


Timothy Feinstein, Ph.D.

> On Oct 21, 2019, at 8:06 AM, Sylvie Le Guyader <[hidden email]> wrote:
>
> *****
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> *****
>
> Human, brain and postmortem... This is definitely a tough one.
> I first would try and work at reducing the autofluorescence without staining the sample.
> For example you can try bleaching the sample (e.g. exposing the sections overnight in a hood with a UV lamp). NAD, flavins... usually bleach very nicely. You can also try oxygen peroxide. Once you find a way to reduce autofluorescence, you optimize the staining.
>
> I would guess that paraffine embedding would make things worse but if you need it, from what we have seen Soudan black seems to work well to reduce the fluorescence due to paraffine embedding. However it sounds like MaxBlock is similar.
>
> You could try and use linear unmixing. Sometimes you cannot see your signal but if you manage to exclude the autofluorescence spectrum, you start finding out that your staining has worked a bit. Then it is easier to troubleshoot. You can acquire the 'pure' fluorophore spectrum by immunostaining cell in culture. Then you use this spectrum to unmix your real sample.
>
> Good luck!
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Avi Jacob
> Sent: 19 October 2019 19:05
> To: [hidden email]
> Subject: Problems with old samples and IHC
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Ced039afff00a4a76f95508d7561f21dc%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637072564023226020&amp;sdata=jt40mVbnhKJ1%2BR%2B%2B7so8ZnFj6g%2FCnPemDrLkgq9RRpQ%3D&amp;reserved=0
> Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Ced039afff00a4a76f95508d7561f21dc%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637072564023226020&amp;sdata=UTPPKX96t0tC%2FL%2FN8fjVG1iPIHFQQp7QXwMP0Q%2Fw77Y%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> Hey all,
>
> I need some help troubleshooting an IHC protocol for a lab.
> 99% of what reaches my facility is fresh, or relatively fresh, from TC or small animal experiments.
>
> The samples we are having issues with are from *human brains* that have been in *formalin for a number of years* (some for over a decade or two) and, according to records, the tissues were not prepared immediately, rather some were processed 48h post-mortem.
>
> The IHC has seen little success: we are getting mostly background with little specificity. A couple of samples do seem to have worked, but we cannot rely on one or two slides (out of many dozens). In some samples, even the dapi is not working well.
> Here are some things that were tried by the lab in order to improve the IHC
> protocol:
>
>   - 24 h incubation in paraffin (as part of the paraffin block protocol).
>   - Antigen retrieval with citraconic-anhydride acid (supposed to handle
>   the formalin crossover).
>   - Increased primary antibody conc. to 1:100.
>   - TSA for secondary in order to strengthen the signal
>   - Two different kinds of blocking were tried, both of which are supposed
>   to work in this type of IHC:  CAS Block and  Goat serum
>   - A *MaxBlock kit *is being used to lower the high autofluorescence
>   inherent to these samples. *(Can this itself be blocking the a.b.'s? I
>   never worked with this before and it gives a black tint to all the samples.
>   Supposedly without this, the samples are impossible to image.)*
>
>
> Does anyone have experience with such old samples and have successfully done IHC with multiple colors?
> Any tips or ideas are very much welcome.
>
> Cheers,
> Avi
>
>
> --
> Avi Jacob, Ph.D.
> Head of The Kanbar Light Microscopy Unit Bar-Ilan University, Ramat-Gan 5290002, Israel
>
>
> När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fki.se%2Fmedarbetare%2Fintegritetsskyddspolicy&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Ced039afff00a4a76f95508d7561f21dc%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637072564023226020&amp;sdata=7uLN4L38HV%2BkZmRPnmy7Bf8R3J9VmFFSssSWc11dyeA%3D&amp;reserved=0>.
>
>
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[Cammer, Michael]

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For bleaching, colleagues have suggested the following:

Quench after fixation and before staining.  Try:
1.  Incubate with NaIO4 (21.4 mg/ml in PBS) 15 min.; PBS wash.
2.  NaBH4 (10mg/ml) in PBS approx. 4 repeated washes 15 min. each; NaBH4 should be dry until immediately before use; PBS wash. [More.]
3.  Incubate with glycine; PBS wash.
Screen for autofluorescence before staining.

And the following were posted in 2002 here or at the microscopy listserv:

From: "James F. Sanzo" [hidden email]

There are several other methods you can try that quench the remaining free aldehyde groups left after glutaraldehyde fixation (below). However, if you are not necessarily interested in ultrastructure, why not use paraformaldehyde? Aldehyde induced fluorescence is usually much less of a problem with the PFA. You may also want to think about whether the fluorescence you are seeing is indeed due to aldehydes - or if it could be due to some other cellular constituent like lysolipids. As an alternative, you may also want to consider using the longest wavelength your imaging and fluorophore systems will allow. Try looking at the background fluorescence above around 590nm. It may be all you need to do.

For blocking free aldehydes remaining after fixation:

1. 1% (or 150mM) glycine with 0.1% Tween in pbs. Wash well. (glycine binds to free aldehydes)

2. 50 mM NH4Cl in PBS. Incubate for 15 min at RT. Wash well. (see Bendyan)

3. 0.1% Na borohydride in PBS. Apply while fizzing and incubate 3 x 10 min on ice. Wash well. (reduces carbonyls)

4. 0.15 M ethanolamine, pH 7.5. Incubate 30 min on ice. Wash well.

Also...

From: Scott Snyder [hidden email]
Subject: Re: Sodiumborohydride and aldehydefixed cells

Probably the best way I have found of getting rid of aldehyde based fluorescence is to get rid of the aldehydes. I often do this by using the bifunctional crosslinking reagent dithio bis(succinimidyl propionate), or DSP for short. This gives comparable structure retention in my hands and essentially no added fluorescence. A good reference for this is Safieko-Mroczka and Bell, Journal of Histochemistry and Cytochemistry v44 No. 6 641-656 (1996).


Cheers-
Michael Cammer


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Monday, October 21, 2019 8:06 AM
To: [hidden email]
Subject: Re: Problems with old samples and IHC

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*****

Human, brain and postmortem... This is definitely a tough one.
I first would try and work at reducing the autofluorescence without staining the sample.
For example you can try bleaching the sample (e.g. exposing the sections overnight in a hood with a UV lamp). NAD, flavins... usually bleach very nicely. You can also try oxygen peroxide. Once you find a way to reduce autofluorescence, you optimize the staining.

I would guess that paraffine embedding would make things worse but if you need it, from what we have seen Soudan black seems to work well to reduce the fluorescence due to paraffine embedding. However it sounds like MaxBlock is similar.

You could try and use linear unmixing. Sometimes you cannot see your signal but if you manage to exclude the autofluorescence spectrum, you start finding out that your staining has worked a bit. Then it is easier to troubleshoot. You can acquire the 'pure' fluorophore spectrum by immunostaining cell in culture. Then you use this spectrum to unmix your real sample.

Good luck!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Avi Jacob
Sent: 19 October 2019 19:05
To: [hidden email]
Subject: Problems with old samples and IHC

*****
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*****

Hey all,

I need some help troubleshooting an IHC protocol for a lab.
99% of what reaches my facility is fresh, or relatively fresh, from TC or small animal experiments.

The samples we are having issues with are from *human brains* that have been in *formalin for a number of years* (some for over a decade or two) and, according to records, the tissues were not prepared immediately, rather some were processed 48h post-mortem.

The IHC has seen little success: we are getting mostly background with little specificity. A couple of samples do seem to have worked, but we cannot rely on one or two slides (out of many dozens). In some samples, even the dapi is not working well.
Here are some things that were tried by the lab in order to improve the IHC
protocol:

   - 24 h incubation in paraffin (as part of the paraffin block protocol).
   - Antigen retrieval with citraconic-anhydride acid (supposed to handle
   the formalin crossover).
   - Increased primary antibody conc. to 1:100.
   - TSA for secondary in order to strengthen the signal
   - Two different kinds of blocking were tried, both of which are supposed
   to work in this type of IHC:  CAS Block and  Goat serum
   - A *MaxBlock kit *is being used to lower the high autofluorescence
   inherent to these samples. *(Can this itself be blocking the a.b.'s? I
   never worked with this before and it gives a black tint to all the samples.
   Supposedly without this, the samples are impossible to image.)*


Does anyone have experience with such old samples and have successfully done IHC with multiple colors?
Any tips or ideas are very much welcome.

Cheers,
Avi


--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit Bar-Ilan University, Ramat-Gan 5290002, Israel


När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_medarbetare_integritetsskyddspolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=9TIEoUTjGed_Z1liNHqGqvBPyCSipP_nV9Awrek6FRE&s=iGZ-lJL3RNrZ7yT7IdDQG-6W8sJU2Qzt8p2MqCKnnBQ&e= >.


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