FREQUENTLY ASKED QUESTIONS for NEUROSTRUCTURAL ANALYTICS

How should the brain tissue be prepared?

​One of the great advantages of our Golgi impregnation method is that we do not need fresh tissue.  Moreover, we can use brain tissue which has been in fixative for several (and, occasionally, many) months. 

The ideal fixative is commercial grade 10% neutral buffered formalin.  Brains can be immersion-fixed or perfused.  We get good staining with both.  We can also use tissue that is fixed with 4% paraformaldehyde.  Please be sure that the formalin fixative is not past its expiration date.
 
Please note that we do not use a Golgi staining “kit.”  Many years of experience with Golgi staining has taught me that many factors can influence the success of the Golgi impregration method.  These would particularly include (but are not limited to): age of the subject, cell population selected for analysis, and length of time in fixative, and length of time in the various staining solutions..  Achieving good Golgi staining is both a science and an art – but simply using a cookbook approach will not always result in successful artifact free impregataions.
The adage “Garbage In, Garbage Out” can particularly apply to Golgi studies. An inadequate or less-than-optimal impregnation can result in structural artifacts which, if not recognized, can result in the generation of incorrect branching and/or spine data.  Experience in the use of this technique is a critical component of its success !

I  presently have fixed brain tissue sitting in formalin; can it also be used?

Generally, yes…but….We can routinely get good staining from brain tissue that has been sitting in formalin for up to about 6 months….longer than that, and the staining can become somewhat more inconsistent (but it still may be do-able…test staining will determine the efficacy of this older tissue).   We almost always run test stains first to assess optimal staining times for the cell population being evaluated.  We have, on occasion, been able to get good Golgi staining of human tissue sitting in formalin for several years, but due to changes in the pH of the fixative this is more challenging.

​I have tissue blocks that are currently embedded in paraffin; can they be used for Golgi staining?

Sorry….this tissue is not amenable for Golgi staining.  Removing the paraffin would require exposing the blocks to chemicals which would preclude good Golgi impregnation. (Some have tried, and have even published data from this source material, but the staining is quite poor and there are lots of artifacts…I wouldn’t trust it.)

I have brains which have already been sectioned; can you use this for Golgi studies?

Yes…but there are additional caveats.  Usually we cut our tissue sections fairly thick (120um).  (Our tissue is embedded in nitrocellulose). That way we can carry out both branching and spine studies on the same sections. Also, our typical technique is to stain entire blocks using our rapid Golgi variant; single section staining is do-able but does not stain as well and is more time consuming.  Also, most pre-sectioned tissue we have received is cut much thinner than our standard and cannot be used for branching studies.  However, we have used the Golgi stained tissue sections (generally prepared from a Golgi staining kit) primarily for dendritic spine analysis without any significant problems.  (And, of course, the cost is adjusted accordingly).

I have frozen tissue; can you use this for Golgi studies?

Sorry…but no ! In our hands, previously frozen tissue does not typically stain well.  This appears to be due to the occurrence of ice crystals (which can occur either during freezing or defrosting stages) and which damages cell membranes.  Damage to cell membranes is a major factor in precluding good Golgi impregnations.

Neurostructural has completed our initial Golgi study and we have subsequently decided to evaluate a second cell population.  Can we do this?

Yes…one major advantage of Golgi staining is that once our slides have been prepared, they are typically stable for many months and even years! So we can go back to those slides time and time again to do analyses of additional cell populations from those slides.

I do not have complete brains available.  Can we still work with NeuroStructural?

Sure….we often receive entire brains, but just as frequently there is only one hemisphere available from each subject.  Occasionally, we may only get coronal blocks of tissue; this, too, is acceptable for our studies (as long as the blocks are at least 2mm thick).

At the completion of the study, are the slides mine?

Absolutely… I will be happy to send any remaining formalin-fixed tissue and all the  slides back to you. They are yours.

We have some Golgi stained slides but are uncertain how generate data to perform the analyses and do statistical analyses or interpret the findings.  Can you help us out?

Happy to…. It is not unusual for colleagues to contact us with Golgi-stained slides that had been prepared (sometimes with a Golgi kit) but are uncertain how to proceed further.  We’ll be happy to look at your slides and decide on the best course of action and work with you to bring the study to fruition.

What dictates the size of the proposed studies?  Is it publishable?

The typical study size that I recommend (e.g., 5-6 brains per group with corresponding analysis of 6-8 neurons per brain from a particular population) is highly publishable.  Of course, we can always further increase the size of the study if requested.  If the study is published I would also hope to be included as a co-author on the study.

I am not in the United States.  Can fixed tissue be shipped to NeuroStructural Analytics?

Absolutely ! We frequently get tissue shipped from countries worldwide.  Sending formalin-fixed tissue for research purposes using a standard carrier (e.g., DHL, FedEx, etc…) has not presented any problems with US customs. ​

The staining just didn’t turn out well enough to conduct the study.  How much do we have to pay?

Nothing!! If the staining doesn’t work, there is no charge. (Fortunately, this is a very rare occurrence).

If you have any additional questions, please contact us.
Ron Mervis, M.S., PhD, at RonMervis@neurostructural.org

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