One more feature: when using DAB products (brown or Black) one needs to select the color of the counterstain that is on the opposite side of the spectrum: DAB alone - methyl green is optimal (the hematoxylin or other blue stains can be slightly purple making the distinction difficult; for NiDAB as I mentioned, the RED counterstains are optimal and work wonderfully - giving as clear cellular detail as hematoxylin. It is not acidity but "reducing agents" that cause NiDAB to fade.

I am not very sure about brain tissues, perhaps you could try using Hematoxylin as a counterstain?

You can use Mayer`s hematoxylin to counterstain along with a blueing reagent such as Ammonia water. It will turn the violet into blue colour.

we do nickel enhanced all the time on our human brains and use neutral red (I can send the protocol if you like). typically you pair brown with blue (hemaxotylin, or nissl) and black with red (neutral red)- that promotes the best contrast for viewing.

We routinely use hematoxylin for counterstain and find that works well.

I my experience Mayer's hematoxylin works good. Depending on how dark a counterstain you need you adjust the stain incubation time.

We always use neutral red for nickel DAB (bluich black) and cresyl violet or haemalaun/haematoxylin for DAB (brown) You could also use the Vector stains, red or green, but I prefer neutral red.

If you just want a nuclear counterstain you can use methyl green. It is extremely easy and time efficient to use.

Try DAPI for nucleus

I agree with Simantini - methyl green is an excellent counterstain for DAB and allows the smallest amounts of deposit to be seen, which can sometimes be obscured with hematoxylin stains.

We used a thionin, nissil stain on 4% para-formaldehyde fixed brain section, (cut frozen sections), to give a pale blue -purple stain to the cells , yet the DAB label was still nicely visible.

Just Hematoxylin is best.

Try neutral red

Hematoxylin

I suggest you may try methyl green for counterstaing nuclear and light green for non-nuclear staining.

We routinely use neutral red to counterstain Nickel DAB in brain.

Hematoxylin. This is the routine counterstain for all IHC with DAB...

I agree with Barbara, thionin is a very good choise.

All of these work well, but Methyl green gives the best contrast in my experience.

10 sec Mayers acid Hemalum and bluing in Tabwater afterward; Toluidine Blue might also work...

Gill II

I would try the thionin nissl stain, that is what we used on brain tissue already stained with DAB and we did not loose the DAB signal

Try to do neutral red 1%

Hematoxylin is the routine counterstain for IHQ. But first, you have to set the staining time to equalize DAB signal and background. The staining time depends on how many times the solution was used. A wash in running water is sufficient to block the reaction of the stain with tissue.

Nuclear Fast red (0.1g NFR in 5g of Aluminium sulphate, boil to dissolve), may be used as counterstain if Haematoxylin does not offer good contrast between brown/black DAB.

I didn't think that any chemical would remove DAB (without Nickel) from sections.

Have you tried doing the Cresyl Violet first and then immuno?

In my experience Mayer's Hematoxylin worked very well, but you have ti be extremely careful in the time. A very short incubation expecially if the haematoxylin gas been prepared immediately before the use is enough.

Any formula of hematoxylin works well, and you can use oil based mount for DAB.

Agree with Karen that methyl green is a good choice.

I think that Haematoxylin is best but stain only for a short time (30s is often adequate with a good haematoxylin). If over-staining occurs it can be removed with a few dips in acid-alcohol (1% HCL in 70% ethanol) followed by "blueing up" either in tap water or ammonia water. In my experience this does not diminish the DAB stain. I do not use nickel enhancement as I have found it to make the coloured deposit darker (and hence closer to haematoxylin in colour) without any increase in signal. Haematoxylin counterstains have the advantage over others in that they provide a good picture of the morphology of the nucleus as well, often allowing identification of specific cell types.

Hi, In our laboratory we never differentiate the hamatoxylin as we have found acid alcohol will readily remove the DAB signal. I believe it is the acid causing your loss of signal. Routinely we use Harris' haematoxylin for 30 seconds. Alternatively use 1% aqueous fast green,1% light green or 1% neutral red. We would blot dry then a quick dehydration through ethanol to xylene then mount in DPX. We have never experienced lost or reduced DAB signal using these counterstain methods.

Hi Jackie, I find it interesting that you find signal loss with acid alcohol. I tend to stain progressively and will not use acid alcohol until absolutely necessary. However, when using Sigma DAB (we buy in the powder and reconstitute ourselves) we have not had any signal loss after a few dips in acid alcohol. However, I will bear in mind what you have experienced. Thanks!

You could try nuclear fast red. We use this on our non brown stained samples.

Kimberley, a good haematoxylin (we currently use either Mayers or Gills) is excellent for looking at Neuroanatomy.

We also use DAB tablets from sigma and haematoxylin from Merck. Try to go with pre prepared kit if your signal is not stable.

A drop per paraffin section for 1 min washed under agitation 3x 30 seconds in 70% etOH is working fine give a nice blue staining for hematoxylin.

In general, I use hematoxylin to countstain the neuclei, but on occasions it enhance the DAB color in the image leading to false positive results. I recommend to use methylene green to countstain.

Use H&E or just one of them (counterstain lightly). May omit H if DAB signals are in nuclei.

I would do Eosin staining . The pink color give good contrast with dark gray of BrdU color.

I agree with other comments. Usually just a light counterstain with haematoxilin is enough. I have seen slides counterstained with fast red and sometimes the staining overlap with DAB (if signal intensity is low)

In my experience Hemat oxilin is the best counterstain when working with DAB. I agree with Jill McMahon!

for the last 23 years we use mayer's hematoxlyine and it works great for 1-2 minutes but if your marker is speicifc for the neuclear antigen such as virus and proteins hormones stain with hematoxyline for 30 seconds and blue in runing water for 1-2 mintues and coverslips with DPX

The fading of NiDAB is due to one of two features. 1) if you keep ANY bleach in the room, the slightest exposure of a slide or glassware or air with some bleach will cause NiDAB to fade (we do not allow ANY bleach in our laboratory but let others nearby keep it. 2) NiDAB will fade if beakers, slides, or any other glassware used in the staining came into contact with Alconox washing solution. NEVER NEVER use the powdered glasswashing material if you plan on using NiDAB. Instead, the liquid diswasher material cleans just as well but keeps the NiDAB from fading. A third consideration is seen only when NiDAB is used in concert with immunofluorescence. While we have conducted many studies with nuclear signals stained with NiDAB (including BRdU) we found that water soluble media cannot be used but instead, the NiDAB and fluorescence are maintained for YEARS when slides are dehydrated and coverslipped with xylene based mounting media (low fluorescent Permount for example, or Krystalon). Then you get the best of both worlds: maintenance of the NiDAB signal and long term storage of the fluorescence. With BRdU this approach in neurons enabled good neuron nuclear labeling for cells not containing the BRdU, and for beautiful BRdU. I do not like cresyl violet but would recommend another stain, Neutral Red which gives much better contrast to the NiDAB product.

One more feature: when using DAB products (brown or Black) one needs to select the color of the counterstain that is on the opposite side of the spectrum: DAB alone - methyl green is optimal (the hematoxylin or other blue stains can be slightly purple making the distinction difficult; for NiDAB as I mentioned, the RED counterstains are optimal and work wonderfully - giving as clear cellular detail as hematoxylin. It is not acidity but "reducing agents" that cause NiDAB to fade.

I Did IHC stain in Brain tissues. I used Mayers Hematooxylin for 2-3 munites as Counter Stain and you can mount it with DPX. If you want to look at virus antigens in the brain cell then counterstain with Harris Hematooxylin

If you will use DAB as chromogen, hematoxylin is a good option.

I think Gill's hematoxylin is a better counter stain than Mayer's hematoxylin

We usually use mayer's hematoxlyine and it works great for 30s, after then wash destilaed water 10 s, 3% amonium water 10s and finally tap water 10s

HI,

Hematoxylin is the routine counterstain

After the DAB staining (10-15 mint), Harris’s Haematoxylin need to stain only 10 sec, then wash in running tap water from reverse side. Finally rinse in PBS for 5 minutes. Dehydrate (70%, 90% and 100% for 15 sec each) and mount the slide.

I think you should always make some kind of trial before deciding the colors that you are going to use.

Chromogens:

From my experience the regular 3,3'-Diaminobenzidine (DAB) gives different kinds of brown depending on the brand that you are using. I recommend Novocastra DAB [RE7190-CE] + Novocastra DAB Enhancer (copper sulfate) [RE7125-CE] for day-to-day immunostaining. If you really need to stain your slides in gray/black then I would recommend to add the Vector Nickel Solution (only the nickel, not the DAB) [SK-4100].

Counterstains that I prefer:

Brown DAB - Gill's Hematoxilin [SIGMA GHS1128] - always use warm tap water to produce a light sky-blue;

Gray/Black DAB - Nuclear Fast Red [SIGMA 60700] - very light.

I didn't like the contrast with DAB and hematoxylin. I found better contrast and more sensitivity when I developed antibody-stained sections with NovaRed (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin (Vector

Laboratories, Burlingame, CA). Vector's hematoxylin works really well and is in a drop bottle. One drop on each section for NO MORE than 3 seconds and washed quickly thereafter gives very sharp images. Low amounts of whatever you are staining for can be picked up easily under the microscope because of the red/blue contrast.

I would try methyl green (what would help is to purify methyl green first with chloroform from a possible contamination by methyl violet) if you are not happy with haematoxylins.

I regularly use Vector Hematoxyline QS ( cat# H-3404) as counterstain with DAB (from vector cat# SK4100) for immunostaining in Kidney, prostate and Lung tissues. It gives excellent contrast. With DAB-Ni if one doesn't like hemotoxiliyn try nuclear fast red which will give better contrast than methyl green.

We always counterstain our IHC brains with methyl green/alcian blue; it's a good alternative to hematoxylin. Definitely make sure to extract the methyl green with chloroform before use, and filter it to avoid chunkiness.

Methyl green is definetely a good option since it contrasts with both purplish and brownish DAB staining.

Hi Kimberly

Have you tried haematoxylene as a counter stain? It may work , although it may sometimes obscure some ihc nuclear targets.

Depending on how much you need to see with the counterstain, neutral red is often useful because it's not as intense as thionin or cresyl violet, but provides at least some staining for cytoarchitecture. Thionin might also be worth trying. Several thionin staining protocols do not require any acetic acid, so if acidity is the concern, that might help.

I've used methyl green with great results. Thionin is typical in the brain, as is hematoxylin - but as others mentioned, that may obscure your target depending on the staining pattern.

I use Mayer's hematoxylin as a counterstain for DAB. I keep the hematoxylin light blue.

Good luck

Agnes

I use Mayer's Hematoxilyn and depending on the protocol, Tissue is stained between one and five minutes. Good luck!

Have you tried toluidine blue or hematoxylin and eosin?

DAB alone is incredibly stable and I have used all of the above, hematoxylin/eosin is a bit risky since light brown and pink do not distinguish that well (Cresyl violet for DAB is OK but with nickel the colors do not offer as good a separation as I like. I suspect in a cresyl violet preparation, if any reducing agents are present, nickel DAB will fade (we saw that with 1) antifade agents when fluorescence were used in immunofluroescent/peroxidase-based double labeling, or 2) when anything that came into contact with the tissue had been washed with alconox! I have banned use of powdered soaps in my laboratory or in glassware washers for that reason. Otherwise, the NiDAB does not fade in my experience provided the dehydration is complete. I like methyl green as a counterstain for DAB (or NiDAB) it is beautifully aqua and the darker DAB is easily separated!

I had the same problem (and it doesn't matter if the stain is DAB or NiDAB. The best way around this is to use NeuN and essentially double label the sections. For mice Chemicon sells a biotinylated version that we use at 1:30,000 and then apply a goat anti-biotin (1:100,000) then stain with DAB rather than Nickel DAB. Use of just streptavidin fluorophore to give a fluorescent counterstain is OK but would require much much more anti-NeuN (probably around 1:1000-3,000).

The acid treatment with BrDU is the problem. I too saw this. What we do is in species other than mice, double label the sections with NeuN (high titer, monoclonal antibody approximately 1: 50,000-1:70,000). In mice, Chemicon has a biotinylated anti-Neun. That antibody is used at 1:20,000-1:30,000 and we stain it by treating the sections after anti-NeuN with an anti-biotin made in goat (1:100,000) overnight, then use ABC with DAB as if the anti-biotin is the primary: biotinylated anti goat, ABC, etc. It is beautiful!. Note those concentrations are best used if you incubate sections in the various primary antibodies for 48 hr. for 24 hr at room temperature, the concentrations needed may be slightly higher.

Oops thought the first comment did not enter properly... Forgive me!

Mayer's haematoxlyine for a minutes is perfect and blue in ruining water for 2 minutes

In rereading your note, the fact that the NiDAB fades is due to one of two features: 1) any antioxidant in the solution fades NiDAB (that means if you washed any of the glassware with Alconox as a detergent, the NiDAB is not stable; 2) if you have bleach in the laboratory, the NiDAB can fade even if it does not come into direct contact with the sections (it is volatile). When none of the above conditions is present, then the NiDAB, if sections are properly dehydrated and cleared etc, will not fade even for more than 20 years. Any water left in the tissue will cause fading eventually. NONE of the above applies for DAB but of course, the Nissl counterstain is then the only option unless you use amplified (TSA) fluorescence and then, of course you must clear the sections with organic solvents (xylenes) and use xylene-soluble mounting media. Properly cleared neither of the products will fade.

As others mentioned, hematoxylin and methyl green both are good as counterstain in IHC staining. But I think the methyl green working better.

I usually use hematoxylin, and I know that definitely eosin is not a good contestant for brown color of DAB.

When using DAB, (not Ni-DAB), Giemsa is a good counterstain, which at the same time intensified the brown color of DAB

When I did my IHC of brain tissue (paraffin section), I used DAB and haematoxylin.

Here are some useful protocol.

http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf

try aqueous eosin if you want to mount with glycerine/glycerine jelly or try orange G in 70% ethanol (if you can dehydrate the tissue) or acid fuchsin.

If you want to use haematoxylin, use Harris haematoxylin which will not colour the extra-nuclear tissue components darkly.

Dr. Hoffman definitely has a great knowhow about this, so follow her advice. But, if you don`t have the mentioned counterstain reagents, and you said, the purpose is only a general recognition of surrounding areas, try to reduce the exposure time in cresyl violet and ethanol.

Whatever stain you use, perform a progressive stain. The acid and alcohol used in regressive counterstaining will ruin the DAB.

I use  Methyl green (Sigma cat. No. M1884) . prepare 1% in water and stain the slides for 15 seconds will give a good contrast  with DAB

Gerald Waneck suggestion is very reasonable i.e. using progressive staining with whatever stain you are using.

With DAB without added Nickel salts, methyl green is my favorite.  With black DAB (nickel salts added) neutral red is wonderful.  I would avoid cresyl violet if staining for DAB alone is not very dark as the stain can sometimes shift to purple and be difficult to separate from the ICC product.  Unfortunately this is the favorite of the pathology community.  If the DAB is strong it is OK, if not, no you obscure the immunocytochemical product.

As a pathologist, I've always used hematoxylin no matter what DAB chromogen was applied.  Tissue (nuclear) morphology stands out well so that one can evaluate tissue changes as well as antigel localization.  

I've recently begun piloting a NovaRED/NiDAB double labeling protocol. Preliminary results are promising. 

Orange -G or Harris Haematoxylin can be tried as counter stain to DAB.