3,3-Diaminobenzidine tetra HCl dehydrate is a stain widely used in histochemistry (HC) and immunohistochemistry (IHC) procedures. 3,3-Diaminobenzidine tetra HCl dehydrate can be used for peroxisome staining and mitochondrial cytochrome c oxidase activity staining.

Physicochemical Properties

Molecular Formula	C12H22CL4N4O2
Molecular Weight	214.2664
Exact Mass	394.049
CAS #	167684-17-5
Related CAS #	DAB tetrahydrochloride;7411-49-6
PubChem CID	2829715
Appearance	Typically exists as solid at room temperature
Boiling Point	481.7ºC at 760 mmHg
Melting Point	280 °C
Flash Point	282.7ºC
Vapour Pressure	1.95E-09mmHg at 25°C
LogP	7.086
Hydrogen Bond Donor Count	10
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	1
Heavy Atom Count	22
Complexity	209
Defined Atom Stereocenter Count	0
SMILES	N([H])([H])C1=C(C([H])=C([H])C(=C1[H])C1C([H])=C([H])C(=C(C=1[H])N([H])[H])N([H])[H])N([H])[H]
InChi Key	BXIYIGBKSGVJOS-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H14N4.4ClH.2H2O/c13-9-3-1-7(5-11(9)15)8-2-4-10(14)12(16)6-8;;;;;;/h1-6H,13-16H2;4*1H;2*1H2
Chemical Name	4-(3,4-diaminophenyl)benzene-1,2-diamine;dihydrate;tetrahydrochloride
Synonyms	167684-17-5; 3,3'-Diaminobenzidine tetrahydrochloride dihydrate; (1,1'-Biphenyl)-3,3',4,4'-tetramine tetrahydrochloride dihydrate; UNII-Z3KH58591V; Z3KH58591V; [1,1'-Biphenyl]-3,3',4,4'-tetramine, hydrochloride, hydrate (1:4:2); (1,1'-Biphenyl)-3,3',4,4'-tetramine, tetrahydrochloride, dihydrate; (1,1'-Biphenyl)-3,3',4,4'-tetramine, hydrochloride, hydrate (1:4:2);
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Biochemical Assay Reagent
ln Vitro	3,3-Diaminobenzidine tetrahydrochloride dehydrate (DAB) is a staining agent that, upon oxidation, forms an insoluble polymer and deposits as a dark brown precipitate at the reaction site, thereby visualizing target molecules. In immunohistochemical applications, this compound serves as a substrate for peroxidase conjugated to primary or secondary antibodies. Its histochemical staining technique is primarily used in the following areas: organelle staining of peroxisomes and activity detection of mitochondrial cytochrome c oxidase (COX, also known as respiratory complex IV) [1]. Guidelines [1] (The following is a recommended protocol and can be adjusted according to specific needs): Cytochrome c oxidase (COX) histochemical staining procedure: 1. Incubate the sections with approximately 50 µL of COX staining solution (containing 0.1 M NaPO₄ buffer, 4 mM 3,3-diaminobenzidine tetrahydrochloride dehydrate, 100 µM cytochrome c, and 2 mg/mL catalase) at 37°C for 45 minutes. 2. Rinse three times with PBS buffer, 5 minutes each, followed by graded ethanol dehydration (75% and 95% ethanol for 1 minute each, and 100% ethanol for 10 minutes). 3. After staining, allow the sample to dry completely before further processing.
References	[1]. Dölle C, et al. 3,3'-Diaminobenzidine staining interferes with PCR-based DNA analysis. Sci Rep. 2018 Jan 19;8(1):1272.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.6670 mL	23.3350 mL	46.6701 mL
	5 mM	0.9334 mL	4.6670 mL	9.3340 mL
	10 mM	0.4667 mL	2.3335 mL	4.6670 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.