Tyrosine Hydroxylase Antibody

Tyrosine Hydroxylase Antibody
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Price: $400.00
Product ID : 22941

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Description

The ImmunoStar monoclonal Tyrosine Hydroxylase antiserum was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat catecholamine neuron systems using indirect immunofluorescent and biotin/avidin-HRP techniques. Recommended primary dilution is 1/4000 - 1/8000 with the biotin/avidin-HRP technique.  This antibody has been used successfully for IHC, ICC, FC, and IP.

Western Blot:  This antibody does not cross react with dihydropterdine reductase, dopamine-B-hydroxylase, phenylethanolamine-N-methyltransferase, phenylalanine hydroxylase or tryptophan hydroxylase using Western Blot methods. Click here for more Western Blot protocol information.

The TH antibody identifies dopaminergic axons and nerve ending during early mouse, rat and human development. This is because tyrosine hydroxylase is expressed very early in the developing dopamine neuron phenotype.

Photo Description: High magnification (above) and low magnification (below) IHC image of the rat midbrain staining for tyrosine hydroxylase. The tissue was fixed with 4% formaldehyde in phosphate buffer, before being removed and prepared for vibratome sectioning. Floating sections were incubated at RT in 10% rabbit serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:5000 for 48 hours, rabbit anti-mouse secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen. 

Tyrosine Hydroxylase midbrain #22941

Host: Mouse

Quantity / Volume: 100 µL

State: Lyophilized Whole Serum

Reacts With: Alpaca (Llama), Amphibian, Anuran (Frog), Anuran (Urodele), Aplysia Californica (Sea Slug), Bee, Bird, Blowfly, Boar, Budgerigars, Canary, Cat, Chick, Chicken, Clam, Cockroach, Cod (Fish), Crab, Crayfish, Dog, Drosophila (Fly), Eel, Emu, Enegal Bichirs (Eel), Ewe, Ferret, Fish, Fly, Fox, Frog (Xenopus Laevis), Fruit Fly, Gastropod (Slug), Gastropoda, Gecko, Gerbil, Goldfish, Guinea Pig, Guppies, Hamster (Mesocricetus Auratus), Helisoma Duryi (Snail), Hen, Human, Iguana, Ilyanassa Obsoleta (Sea Snail), Lamprey, Leech, Lizard, Lobster, Locust, Mexican Axolotl (Salamander), Mollusca, Monkey (Macaca Fascicularis), Mouse, Newt, Opossum, Parrot, Periplaneta Americana (Cockroach), Phestilla Sibogae (Sea Coral), Phestilla Sibogae (Sea Slug), Phoronis Pallida (Phoronida), Pig, Pigeon, Possum, Prawn, Quail, Rabbit, Rat, Renilla Koellikeri (Sea Pansy), Rodent, Salamander, Salmon, Sea Fish, Sea Slug, Shark, Sheep, Short-Beaked Echidna, Shrew, Snail, Snake, Sparrow, Spisula (Clam), Squirrel Monkey, Starling, Stickleback, Tadpole, Trout, Turkey, Turtle, Urodele Amphibian, Water Buffalo, Worm, Yeast, Zebra Finch, Zebrafish

Availability: In Stock

Alternate Names:  Tyrosine 3-monooxygenase (TH); TYH; Tyrosine 3-hydroxylase, anti-TH, anti-tyrosine hydroxylase

Gene Symbol / ID, Accession #:  Th, 25085

Reviews

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Reviewed by mdreed
10/03/2016 - 02:18:02 PM
Wonderful Antibody!

This antibody produced excellent staining in bullfrog (Lithobates catesbeianus) brainstem using both Ni-DAB and IF. Staining was extremely clear with very little background at a concentration of 1:1000.

Tissue Preparation
Brainstem was removed from bullfrog and placed in 4% Paraformaldehyde overnight, then transferred to a 30% sucrose solution for at least 4 hours (until brain sinks). Brain was then placed in embedding media and flash frozen in hexane solution at -80 C. Brains were sliced to slides on a microtome to 20um. Slides were allowed to dry overnight prior to staining procedure.

For DAB Staining
Day 1
1. PBS 1X wash (3X5min)
2. H2O2 incubation 30min (room temperature)
3. PBS 1X wash (6X5min)
4. Blocking Buffer 1-2hr (PBS 1X+Normal serum+0.3% Triton 100X)
5. Primary antibody incubation overnight (PBS 1X+Normal serum+ 0.3% Triton 100X, Room Temperature)
Day 2
1. PBS 1X wash (5X5min)
2. Biotinylated secondary antibody incubation 1-2hr (PBS 1X+Normal Serum, Room Temperature)
3. PBS 1X wash (4X10min)
4. Incubate in ABC solution 1hr (Room Temp)
5. PBS 1X wash (6X5min)
6. Sodium Acetate wash (3X5min)
7. Ni-Sulfate DAB incubation 8min
8. 1-2 min H2O2 on Ni-DAB
9. Sodium Acetate wash (3X5min)
10. PBS 1X wash (3X5min)

This protocol worked great for TH staining in bullfrog and has been used for 2 years from frozen aliquots with great results.

Mitchell Reed, PhD Candidate
Department of Biology and Wildlife
University of Alaska-Fairbanks

Reviewed by user
04/01/2016 - 11:33:47 AM
Antibody performed very well

The tyrosine hydroxylase antibody was successfully used for immunostaining of dopamine neurons in the adult Drosophila brain. The antibody performed very well for this application.

Drosophila brains were collected and stained as follows:
1. Dissected in PBT buffer
2. Fixed in 4% PFA for 20 min at RT
3. Washed in PBT twice
4. Blocked in 5% normal goat serum in PBT buffer for 30 min at RT
5. Incubated in anti-TH primary (1:1000) for 48 h at 4 deg C
6. Washed 5 times in PBT
7. Incubated in anti-mouse alexa fluor-488 secondary antibody for 48h at 4 deg C
8. Washed 5 times in PBT before mounting and imaging

Dopamine neuron cell bodies and projections within the canonical anterior and posterior clusters could be readily visualized by confocal microscopy z-stack imaging. There was very little background staining in the Drosophila brain.


Submitted by:

Ian Martin, PhD
Jungers Center for Neurosciences Research
Parkinson Center of Oregon
Oregon Health and Science University

Reviewed by user
11/17/2015 - 11:00:28 AM
Consistent Results

I have used the mouse antibody for tyrosine hydroxylase (Product ID : 22941) for staining dopaminergic cells in Drosophila melanogaster adult brains, larval brains, as well as primary neuronal cultures, and have always had consistent results, as well as a good signal-to-noise ratio of 4 to 1.

In published results, I compared the staining of this antibody with a GFP signal driven by tyrosine hydroxylase and the overlap between the two signals was good (J Neurochem. 2013 Aug;126(4):529-40. doi: 10.1111/jnc.12228. Epub 2013 Mar 24).

Also, in unpublished results, it appeared to perform just as well as a Drosophila antibody for tyrosine hydroxylase. I have used this antibody for years and have always obtained consistent results.


Lyle Wiemerslage, PhD
Biomedicinska Centrum (BMC)
Uppsala Universitet
Sweden

Reviewed by user
06/29/2015 - 12:51:23 PM
Produces distinct staining with very low background

We have used the ImmunoStar Inc. tyrosine hydroxylase antibody (22941) with great success, for identifying TH-IR cells in the striatum and midbrain of European starling (Sturnus vulgaris). We typically use it at a 1:3000 dilution, which produces distinct staining with very low background.

Tissue preparation:
Birds are transcardially perfused with 1X phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The brain is extracted and post-fixed in 4% paraformaldehyde overnight, then transferred to 30% sucrose for 2-3 days (until the brain sinks). Brains are sectioned at 40 µm on a sliding microtome equipped with a freezing stage, and collected into PBS.

Immunohistochemistry procedure:
Brain sections are transferred to 24-well plates. All solutions are made up in 1X PBS and are typically applied at a volume of 0.5 ml. The plates are gently agitated on a shaker during incubations.

Day 1:
• Rinse in PBS 3 times, for 10 min each
• Incubate in 3% hydrogen peroxide for 10 min
• Rinse in PBS 3 times, for 10 min each
• Incubate in blocking serum (from Vector ABC Elite kit) + 0.2% Triton X-100 for 30 min
• Incubate in primary antibody + 0.5% Triton X-100 overnight at 4°C

Day 2:
• Rinse in PBS 3 times, for 10 min each
• Incubate in secondary antibody (from Vector ABC Elite kit) + 0.6% Triton X-100 for 30 min
• Rinse in PBS 3 times, for 10 min each
• Incubate in ABC reagent (from Vector ABC Elite kit) + 0.6% Triton X-100 for 30 min
• Rinse in PBS 3 times, for 10 min each
• Incubate in 0.05% DAB (3’3-diaminobenzidine) + 0.015% hydrogen peroxide for 2-3 min
• Rinse in PBS 3 times, for 10 min each
Float-mount sections onto gelatin-subbed slides and let dry overnight, then dehydrate with ethanol and xylene rinses and coverslip with Permount.

Joseph M. Casto, Ph.D.
Associate Professor
School of Biological Sciences
Campus Box 4120
Illinois State University
Normal, IL 61790-4120

Reviewed by user
01/29/2015 - 01:32:20 PM
Used this successfully for years

Tissue used: Free floating, 25-40µm thick sections from perfused mice (4% PFA followed by 10% sucrose dehydration).

Protocol steps: Standard IHC protocol using vector biolabs ABC mixture and DAB reagent

Concentration of TH antibody: 1:5000, left overnight in PBS+0.3% triton x-100
Staining results: Specific staining of typical dopaminergic neurons within the midbrain (VTA and SNc). Cytoplasmic staining.

I have used this successfully over the past years (see Rousseaux et al, 2012, PNAS; Aleyasin, Rousseaux et al, 2010, PNAS)

Submitted by:

Maxime Rousseaux, Ph.D.
Laboratory of Dr. Huda Y. Zoghbi
Department of Molecular and Human Genetics
Baylor College of Medicine

Reviewed by cmharley
05/20/2014 - 04:33:05 PM
Perfection

We have gotten truly incredible results with the Immunostar TH antibody. We have been using the antibody to label tissue from several different invertebrates with fantastic results. The confocal images are un-real.

We fix tissue in 4% paraformaldehyde then rinse 3x (15min each) in PBS and place in a block solution overnight. After that we place it in a 1:100 dilution of this primary and leave it alone in the fridge for 4 days. Tissue is then placed in a dilutant solution and in secondary antibody in the fridge at least overnight. We then rinse, dehydrate, and mount.

We have used this protocol for moths, leeches, and spiders without issue.

Cynthia Harley
Department of Entomology
University of Minnesota

Reviewed by goozly
05/15/2014 - 12:09:24 PM
Works Consistently Great on Rat TH

Used it at 1:5000 overnight for immunofluorescence experiment (floating 40um sections) on rat locus coeruleus, and got stunning images. Also worked well in a colocalization experiment when mixed with a primary for glucocorticoid receptor (raised on rabbit).


Lishay Goozy Alaluf
Dept of Biochemistry and Molecular Biology
New York Medical College

Reviewed by BrownLab
05/06/2014 - 05:50:24 PM
Works Great!

I use this antibody regularly to stain for tyrosine hydroxylase in sections of rat retina. I have always been very happy with the results and found that the best working dilution to be 1:1000. It produces a strong image with very little background and I would definitely recommend this antibody to other users.

1. Fixed the tissue in 4% paraformaldehyde and PBS
2. Collected the eye and then sectioned it into 20 micron slices using a cryostat
3. Placed sections in blocking buffer for 1 hour

4. Placed the sections in primaries overnight:
1:5000 dilution of in house anti rat melanopsin
1:1000 dilution of your TH antibody

5. Put the sections in secondary antibodies for 2 hours:
1:2000 alexa fluor 488 dk anti rabbit
1:2000 alexa fluor 594 dk anti mouse

6. Then we imaged it using a max projection of a Zeiss laser scanning confocal.


Emily Gray
Dr. Lane Brown Lab
Head Laboratory Technician
Department of Physiology & Neuroscience
Washington State University

Reviewed by user
06/25/2013 - 02:25:50 PM
Wonderful Results

We have been using the Immunostar anti-tyrosine hydroxylase antibody on Drosophila brain tissue with wonderful results. No background labeling, very bright signal, and worked the very first time with no modifications to our protocol required! We made aliquots of the antibody as per the instructions (dilute 1:10 in PBS for storage in the freezer, then dilute an additional 1:5 for staining) and used the following protocol.

1. dissect brains in saline and fix for 20 minutes in 4% PFA
2. rinse 3x in PBST and place in block solution (5% NGS in PBST) for 20 minutes
3. place in primary antibody (1:50 total dilution in PBST) for 2 days at 4 degrees
4. rinse 3x in PBST
5. place in secondary antibody (1:400) for 2 days at 4 degrees
6. rinse 3x in PBST, then mount tissue on slides

Katherine A Tschida, Postdoctoral Associate
Duke University
Duke Biology
Durham, NC

Reviewed by user
04/01/2013 - 02:04:31 PM
A Fabulous Antibody!

I have routinely used this monoclonal antibody to stain for tyrosine hydroxylase (#22941) in both rat and mouse brains for several years with consistent and excellent results. It is the best antibody out there! Over more than 10 years I have used different lots with equally high quality results. The best part of using this particular antibody is that staining is fabulously strong with high dilution rates up to 20,000x, so one batch can go a very long way. I even sometimes re-use a solution with good results still. Moreover, crisp soma dendrite and axonal staining can be achieved with both immunoperoxidase (using avidin-biotin complex) and immunofluorescence methods. It works great in multi-labeling studies in which I combined this antibody with immunolabeling of c-Fos or other markers.

Section preparation:
Mice and rats are perfused with 4% paraformaldehyde in 0.1M phosphate buffer (PB). Generally I add 15% of saturated picric acid solution, but for this staining it is not required. It may be informative to know that adding up to 0.5% glutaraldehyde does not affect the staining results (thus suitable for EM studies). After each brain is removed, it is post-fixed in 4% paraformaldehyde overnight, then blocked and sliced in 40 or 50 micrometer sections on a vibratome. Sections are collected free-floating in series in multi well plates and stored in 0.1M PB to which 0.1% sodium azide is added for long-term storage at +4°C.

Day 1.
The sections chosen for staining are then pretreated in 0.1% sodium borohydrate (0.1%) in PBS for 10 minutes, and also in 0.3% hydrogen peroxide 0.1% sodium azide mixture in PBS for 30 minutes to quench peroxidase activity. To suppress nonspecific binding sections are left overnight in a mixture of normal serum and Fab fragment of anti mouse IgG (usually goat serum and IgG – if use of goat secondary antibodies are appropriate). All antibody solutions are made up in PBS to which is added 0.1% sodium azide (for repeated uses) and 0.5% Triton X-100 (labeled PBS-T/A).

Days 2-3.
After 3 washes in PBS, sections are incubated for at least 24 h (often 2 days at 4 °C) in anti TH at a dilution of 10,000x in PBS-T/A containing 2% normal goat serum (NGS).

Day 3-4
Following washes in PBS sections are transferred to the secondary antibody (e.g. goat anti-mouse IgG-Alexa488 for or biotinylated goat anti-mouse IgG) in PBS-T/A with 2% NGS. This incubation can last from 4 h (at RT) or overnight at 4°C. For immunofluorescence sections can be mounted and evaluated after this step.

Day 4-5 and on.
Biotinylated secondary antibody incubation is followed by PBS washes, and transfer into Avidin-Biotin complex solution (Vector Labs) diluted 1000 x in PBS-T (no azide added this time!). This incubation lasts from 4 h to overnight at 4°C. After that the sections are rinsed in PBS and Tris-HCl solutions (pH 7.6) and stained using diaminobenzidine. I prefer not to use straight H2O2 but add glucose oxidase (1 microliter per 5 ml) to the DAB mixture and infuse D-glucose (50 ul of 10% solution per 5 ml) into the wells to start the reaction slowly.

Ronald P. Gaykema, Ph.D.
Department of Pharmacology
University of Virginia Health System
Charlottesville, VA 22908

Reviewed by user
02/22/2013 - 10:46:50 AM
Highly Recommend This Antibody

We have used the Tyrosine Hydroxylase antibody #22941 for western blotting on mouse brain lysates. The antibody is specific and gives clean bands. It works both using the ECL and the LICOR Odyssey system using infrared dyes.

I therefore highly recommend this antibody.

Kristina Becanovic, PhD
Department Clinical Neuroscience
Karolinska Institutet
171 76 Stockholm

Reviewed by user
07/16/2012 - 02:41:33 PM
One of the Best Antibodies

We have used this antibody (22941) to detect endogenous levels of TH in mouse and human cells. For cryosections, it works better after antigen retrieval (sodium citrate). The dilution we use is 1:250, and overnight incubation with the primary at 4°C increases the signal quite a lot.

It works beautifully giving a clear strong signal, with low background. One of the best antibodies to detect TH that we have found.


Lottie Jansson Sjostrand
Karolinska Institutet
Stockholm Sweden


Reviewed by user
04/23/2012 - 02:03:41 PM
Highly recommend!

The TH antibody worked very well with no issues or background. I highly recommend this antibody to my colleagues.

Zebra finch brain tissue embedded in gelatin (from perfused birds)
30um sections stored in cryoprotectant
Rinse 6 X 5 min in PBS
Wash 15 min in 0.5% H2O2 in PBS
Block 1 hour in 5% normal donkey serum in PBS with 0.3% TritonX (PBST)
Incubate in TH antibody (1:1,000 dilution) in PBST for 2 days at 4°C.
Rinse 3 X 5 min in PBS.
Incubate 1 hour in secondary antibody.
Rinse 3 X 5 min. in PBS @ RT.
ABC elite kit (Vector)-1 hour
Rinse 3 X 5 min in PBS
DAB plus 0.0075% H2O2 for 7 minutes.
Rinse in PBS.
Mount onto slides, dehydrate and coverslip with DPX


Dr. Juli S. Wade
Dr. Yu-Ping Tang
Neuroscience Program
Michigan State University

03/28/2012 - 10:19:20 AM
Beautiful Labeling Images

We used ImmunoStar Tyrosine Hydroxylase Antibody, Product ID: 22941, to label rat brain C1 region catecholaminergic cells, and got very beautiful labeling images.The protocol we used:

Day1
Select tissue sections (frozen, 35 um);
Rinse sections in PBS for 2x10 min on shaker at room temperature;
Incubate sections in ADS (Antibody dilution solution) for 2 hrs on shaker at room temperature;
Prepare primary antibody solution;
Ms anti-TH (ImmunoStar, Catalog: 22941), use at 1:1500;
Incubate sections in primary antibody solution overnight on shaker at room temperature;

Day2
Rinse sections in T-PBS for 2x10 min on shaker at room temperature;
Prepare secondary antibody solution;
Donkey anti Ms A488 (Molecular Probes, A21202), use at 1:500;
Incubate sections in secondary antibody solution for 1 hr on shaker at room temperature;
Rinse sections in PBS for 2x10 min on shaker at room temperature.
Mount sections on Superfrost Plus Microscope Slide.
Coverslip mounted sections with anti-fade mounting regent.

Rubing Xing, Senior Research Assistant
Neurological Surgery
Oregon Health & Science University

03/01/2012 - 01:27:40 PM
Exceptional Results

I have used your TH monoclonal antibody 22941 for many years with beautiful results. This is one of the antisera we use on an almost daily basis. We are extremely glad that you were able to confirm the specificity of the TH antibody in transfected cells.

Thank you and all the technical staff for the valuable assistance and your wonderful help in its characterization.

Review submitted by:

Virginia M. Pickel, PhD
Division of Neurobiology
Weill Cornell Medical College,

Q & A Wall

Login or Register to ask any question about this product.

We use PFA perfused and sucrose cryoprotected mouse brains to visualise axon growth in dopaminergic cells. I was wondering if your antibody can visualise the axonal tract during early development nicely? We use the antibody routinely (refer Pasterkamp lab, UMC Utrecht) and it would be great if we can test this antibody to see this. Although we have a working Rb polyclonal antibodies we want a different host for costainings and one that can mark axons with same efficiency. We would be very glad to compare and show the staining quality from all TH antibodies we have for your company.
Question By Utrecht, The Netherlands

Yes, this antibody identifies dopaminergic axons and nerve ending during early mouse, rat and human development. This is because tyrosine hydroxylase is expressed very early in the developing dopamine neuron phenotype. In summary, because of the extraordinary affinity of this antibody for tyrosine hydroxylase, if tyrosine hydroxylase is present, this antibody will detect it.

If you want images you will find a number of papers using this antibody to study dopamine neuron development.

Answer By Greg on 2015-10-05 09:22:59

It would be useful to know whether the example blot was run under reducing (with beta mercaptoethanl or DTT) conditions?
Question By St Francis Xavier University

Yes, of course, you must run SDS-PAGE using reducing conditions, I.e., beta-mercaptoethanol or dithio-threitol. Otherwise TH subunits will not completely dissociate and you will see a high molecular weight smear on the Western.

Answer By Tech Support on 2015-05-12 12:49:10

I would like to know can the TH Antibody be used for flow cytometry on fixed cells (only analysis) in indirect immunomarking revealed by a fluorescent second antibody ? - Can it be used for ICC on cultured cells also fixed and revealed like before ? In the datasheet, the antibody is used on frozen section, does the conformation of the epitope changes when frozen? If the tissue is not frozen, will the antibody still recognize the epitope?
Question By Céline

1.Yes, this antibody has been used for flow cytometry of fixed cells.
2.Yes, the principal use is ICC on fixed cells in culture.
3.No, the epitope is not modified by freeze-thaw.
4.Since the antibody can be used for immunoprecipitation it must recognize the native none denatured protein in fresh tissue.


Answer By Tech Support on 2013-06-06 15:34:47

I would to know how is the storage and shipment of 22941. Is 4° C or ice dry?
Question By Celeste

The Tyrosine Hydroxylase Antibody (# 22941) is freeze-dried, so we do not ship it with ice. More information can be found on our FAQ page.

Answer By Colleen Hammer on 2013-05-07 12:26:57

I would like to know if the IMMU-22941 works on paraffin sections as in your data sheet only frozen and vibratome sections are cited. I will use your AB on crab's nervous system but as I do not have criostat or vibratome I must to use paraffin inclusion. I'll use the secondary AB biotin conjugated and streptavidin as cromogen, DAB of course my staining system. There is another information I would like to have the AB concentration in your 100 uL vial.
Question By Silvia

The mouse monoclonal Tyrosine Hydroxylase (# 22941) should work fine in paraffin sections, as long as the tissue is well fixed prior to embedding.

The dilutions we recommend on the specification sheet are based on perfused tissue and vibratome sections. For your paraffins, you should run a dilution series to optimize your application.

Because the TH Antibody is a whole serum (normal mouse serum) we do not know the concentration. Protein concentration is a more meaningful measure for affinity purified antibodies, which are antibodies removed from the whole serum and usually provided in a buffer matrix. If you measure the protein concentration of affinity purified antibodies, you have a measure of how much antibody is in the vial. If you measure the protein concentration of whole serum, you get a number that reflects
all of the protein, not just antibody.

Answer By Tech Support on 2012-07-06 12:23:34

I would like to know some information about your product N°22941, in the data sheet of this antibody there is not information about the epitope used to produce it.
Question By Giovanna

The immunogen used to produce the Tyrosine Hydroxylase antibody (TH) was full-length tyrosine hydroxylase purified to homogeneity from PC12 cells, which are of rat origin. So, TH is directed against full-length rat TH.

The epitope recognized by this antibody is not known but it does not reside within the N-terminus of TH. Previous studies have shown that partial digestion of TH with the protease chymotrypsin removes the N-terminus of TH, producing a C-terminal product of ~ 34 kDa that is catalytically active. Unpublished studies of ours show that partial digestion of rat TH with chymotrypsin and analyzed by western blotting using TH results in an immunoreactive band at 35 kDa.

Based upon this evidence, we conclude that LNC1 recognizes an epitope outside of the N-terminus of the protein and in the C-terminal "catalytic core".



Answer By Technical Support on 2010-05-10 12:33:01

MSDS

This product contains the preservative sodium azide. The concentration percent of the sodium azide is ≤ .09%. Although this hazardous substance is a concentration below that required for the preparation of a Material Safety Data Sheet, we created a standard MSDS for your records.

Download MSDS