Paraformaldehyde-Glutaraldehyde Mixed Fixative (4%/1%) - BioReagent

Cat. No.: P1528808

GRADE & PURITY BioReagent

Storage

Store at 2-8°C

Shipped In

Wet ice

Panoramica

Paraformaldehyde-Glutaraldehyde Mixed Fixative (4%/1%) is a classic aldehyde-based mixed fixative specifically designed for electron microscopy (transmission electron microscopy) sample preparation. This fixative is composed of 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer (pH ~7.4). Its core advantage lies in combining the excellent membrane penetration of paraformaldehyde with the rapid and stable crosslinking capability of glutaraldehyde for protein ultrastructure. The increased glutaraldehyde concentration (compared to the 0.5% formulation) further enhances crosslinking fixation of cellular ultrastructure, making it more suitable for electron microscopy samples requiring high structural preservation. It effectively prevents autolysis and maximally preserves the native state of organelles and subcellular structures. It should be noted that this fixative is primarily directed at protein fixation and is therefore not suitable for fixing nucleic acids or polysaccharides.

Specifications

Specifiche e purezza

BioReagent

Condizioni di conservazione di stoccaggio

Store at 2-8°C

Spedito in

Wet ice

Questo prodotto richiede spedizione a catena fredda. I servizi di terra e altri servizi economici non sono disponibili.

Grado

BioReagent

Product Line

Prodotti biologici

Application Protocols

The following are literature/general protocols for research workflows using 4% formaldehyde + 1% glutaraldehyde fixative. Adjust volumes and times to specimen size and sensitivity.

• Cultured cell fixation (immunofluorescence):

  1. Warm fixative to RT. Remove culture medium and gently rinse cells with PBS.
  2. Add sufficient fixative to cover cells; incubate 10–15 min at RT.
  3. Aspirate fixative; wash 3× with PBS (5 min each).
  4. Quench residual aldehydes with 50 mM NH4Cl or 50–100 mM glycine in PBS for 10 min; wash 2×.
  5. Permeabilize (if needed) with 0.1–0.2% Triton X‑100 in PBS for 5–10 min; proceed to blocking and antibody staining. Note: GA can increase autofluorescence; evaluate need for sodium borohydride quench on robust samples (use caution).

• Tissue sections (10–50 µm):

  1. Equilibrate fixative and samples to desired temperature (RT or 4 °C).
  2. Incubate sections 10–30 min with gentle agitation.
  3. Wash 3–4× with buffer; quench as above; proceed to staining or cryoprotection.

• Small EM tissue blocks (≤1 mm):

  1. Pre‑cool fixative to 4 °C for delicate ultrastructure.
  2. Immerse blocks for 30–90 min with gentle mixing; ensure appropriate buffer/osmolarity.
  3. Rinse thoroughly in EM buffer (e.g., phosphate/cacodylate), then postfix in 1% OsO4 (institutional protocols) and continue dehydration/embedding.

Exact buffer matrix, pH, and stabilizers for this SKU are Not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

This product is a chemical fixative mixture and does not have a native biological role. The following describes its chemical interactions with biomolecules during fixation (literature/general):

• Proteins:
  • Formaldehyde forms methylene bridges between nucleophilic residues (e.g., lysine, arginine, histidine, cysteine), stabilizing tertiary and quaternary structures and immobilizing proteins in situ.
  • Glutaraldehyde, via bifunctional aldehyde groups, yields dense crosslink networks, preserving cytoskeletal and membrane‑associated proteins and improving ultrastructural integrity.
• Nucleic acids:
  • Aldehydes can create crosslinks with nucleobases and protein–DNA complexes, reducing diffusion and preserving nuclear morphology; excessive fixation may reduce accessibility for hybridization unless retrieval steps are applied.
• Lipids/membranes:
  • Aldehydes do not directly polymerize lipids but stabilize lipid–protein interfaces; subsequent osmium tetroxide postfixation (EM workflows) provides additional membrane contrast and stabilization.
• Enzymatic activity:
  • Crosslinking typically inactivates enzymes; activity assays should be performed pre‑fixation or adapted for fixed specimens.
• Autofluorescence:
  • Residual aldehyde adducts and lipofuscin-like products can contribute to background fluorescence, more pronounced with glutaraldehyde. Quenching with glycine, ammonium chloride, or sodium borohydride reduces background.
• Permeabilization interplay:
  • After fixation, detergents (e.g., Triton X‑100, saponin) or alcohols are used to permeabilize membranes for probe access; optimization is specimen‑ and antigen‑dependent.

Note: All uses are for research and laboratory workflows only.

Buffer Applications

Although not a classical pH buffer, this mixed fixative is used in conjunction with physiological buffers to control pH and osmolarity during fixation.

• Common buffer systems during fixation (literature/general):
  • Phosphate‑buffered saline (PBS), pH ~7.2–7.4: Widely used for cells and tissue sections.
  • HEPES buffer (10–25 mM), pH 7.2–7.5: Good buffering capacity around neutrality and minimal metal interactions.
  • EM buffers: 0.1 M sodium phosphate or 0.1 M cacodylate, pH 7.2–7.4, adjusted for osmolarity (e.g., 250–350 mOsm) to minimize ultrastructural artifacts.
• Practical considerations:
  • Avoid primary amine buffers (e.g., Tris) during fixation because they quench aldehydes; reserve Tris for post‑fix washes.
  • Pre‑equilibrate buffer and fixative to the same temperature and osmolarity as the specimen’s medium to minimize shrinkage/swelling.
  • After fixation, wash specimens with the same buffer used for fixation to remove unreacted aldehydes; include a quenching step (e.g., 50–100 mM glycine or 50 mM NH4Cl for 10–20 min) to reduce background.
• Recipes and exact buffer composition for this SKU: Not specified for this item; refer to CoA/Spec Sheet.

If your workflow requires metal‑free or specific ionic conditions (e.g., Ca2+/Mg2+ preservation for junctions), ensure the buffer selection reflects those needs and verify compatibility with downstream stains.

Green Alternatives

While aldehyde fixatives are widely used for sample preservation, they pose health and environmental hazards. Consider greener or lower‑hazard options when compatible with analytical goals.

• Comparative overview (literature/general):
  • 4% PFA only (methanol‑free):
    • Pros: Lower sensitization potential than mixtures containing glutaraldehyde; reduced autofluorescence; often adequate for immunofluorescence.
    • Cons: Weaker ultrastructural preservation vs. GA‑containing mixes.
  • Glyoxal‑based fixatives:
    • Pros: Reported lower toxicity and faster penetration; improved epitope preservation for certain targets.
    • Cons: Different crosslink chemistry; may not match EM‑level ultrastructure; protocol re‑optimization required.
  • Alcohol fixatives (ethanol/methanol):
    • Pros: Non‑crosslinking, rapid dehydration/precipitation; good for nucleic acid preservation in some assays.
    • Cons: Significant shrinkage/antigen loss for some proteins; flammability.
  • Non‑aldehyde crosslinkers (e.g., EDC/NHS, DSG/DTBP):
    • Pros: Targeted chemistries; can reduce autofluorescence.
    • Cons: Cost, handling complexity, and varying penetration; not routine for bulk tissue fixation.
• Environmental/practical considerations:
  • Use minimal volumes consistent with adequate fixation; capture vapors in fume hoods; segregate aldehyde waste for proper treatment.
  • Where GA is essential (EM), reduce concentration and exposure time to the minimum that preserves required features.
• Selection guidance: Choose PFA‑only for fluorescence‑centric studies; use PFA/GA mixes when ultrastructure is paramount. Pilot comparative tests on representative specimens are recommended.

Pharmaceutical Uses

This product is supplied for research use only and is not intended for human or veterinary applications. It is not an excipient or a finished pharmaceutical ingredient.

• Typical role in regulated environments (contextual, literature/general):
  • Aldehyde fixatives may be used in quality-control histology and in‑process characterization of biological materials; however, such uses require facility‑specific validation and do not imply suitability as a pharmaceutical component.
• Regulatory/compendial status:
  • No pharmacopeial monograph is implied for this mixed solution. Any references to formaldehyde or glutaraldehyde monographs do not extend to this specific formulation.
• Formulation roles: Not applicable; aldehyde fixatives are not used as excipients due to their high reactivity and toxicity.

Summary: Limit use to laboratory research and method development in microscopy or analytical characterization. For any GMP‑adjacent use, consult your QA unit and the product’s CoA/SDS; this SKU carries no clinical claim.

Physical Properties

• State/form: Aqueous solution of aldehydes (4% formaldehyde + 1% glutaraldehyde). Appearance and exact buffer/pH: Not specified for this item; refer to CoA/Spec Sheet.
• Concentrations (from Product Name): 4% (w/v) formaldehyde; 1% (w/v) glutaraldehyde.
• Density, refractive index, UV cutoff, metal limits, and stabilizers: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility (general): Completely miscible with water; miscible with many polar organics (e.g., alcohols). Proteinaceous materials are rendered insoluble upon fixation due to crosslinking.
• Volatility/odor (general): Pungent aldehydic odor; vapor release possible from open vessels—use containment.
• Typical pH used for fixation buffers (literature/general): ~7.2–7.4 for light microscopy in phosphate or HEPES buffers; ~7.2–7.4 with controlled osmolarity (e.g., phosphate or cacodylate) for EM. This product’s actual pH is Not specified for this item; refer to CoA/Spec Sheet.
• Component reference properties (literature/general, not item-specific):
  • Formaldehyde (monomer): BP ~–19 °C (gas), typically handled as 37–40% aqueous solution (formalin) with density ~1.08 g/mL at 20 °C; exists mainly as methanediol in water.
  • Glutaraldehyde: Commercial 25% aq. solutions have density ~1.06 g/mL at 20 °C; exists as hydrated/oligomeric forms influencing reactivity.
• Stability notes (general): Aqueous formaldehyde can polymerize upon prolonged storage or at low temperature; glutaraldehyde can self-condense over time. Temperature control and closed containers mitigate changes (see Storage tab).

Quality and Grades

• Grade reported for this item: BioReagent (from Product Data). BioReagent grade typically signifies suitability for common laboratory and life‑science workflows (e.g., cell/tissue fixation), with attention to low particulates and consistent performance. Exact acceptance criteria for this SKU are Not specified for this item; refer to CoA/Spec Sheet.
• Intended use: For research use only (from Product Data). Not for human or veterinary use.
• Stabilizers and additives: Not specified for this item; refer to CoA/Spec Sheet. Some formaldehyde solutions include methanol as a stabilizer; mixed fixatives formulated from paraformaldehyde are often methanol‑free—verify on the CoA if critical for your application.
• Buffer matrix, pH, and osmolarity: Not specified for this item; refer to CoA/Spec Sheet. These parameters directly affect morphology and antigenicity; confirm prior to sensitive EM/IF workflows.
• Lot-specific documentation:
  • Certificate of Analysis (CoA) typically includes concentration verification, appearance, and possibly pH and sterility/bioburden data where applicable. Always consult the lot CoA for release criteria and any in‑process controls.
• Performance notes (general best practice):
  • For immunostaining, methanol-free formaldehyde sources are often preferred to minimize antigen masking.
  • For ultrastructure preservation, the presence of 1% glutaraldehyde supports robust crosslinking, though it may increase autofluorescence; quenching steps are recommended (see Protocols tab).

Reaction and Applications

This mixed fixative is designed for chemical fixation of biological samples, leveraging complementary reactivities of mono‑ and di‑aldehydes to stabilize cellular architecture.

• Mechanistic basis (general chemistry):
  • Formaldehyde reacts primarily with nucleophilic centers (e.g., lysine ε‑amines, N‑termini, cysteine, histidine) to form reversible Schiff bases and more stable methylene bridges upon maturation, providing rapid penetration and uniform protein crosslinking.
  • Glutaraldehyde, as a dialdehyde, forms multiple covalent linkages leading to robust intra-/intermolecular crosslinks that preserve ultrastructure (membranes, organelles) but may increase autofluorescence and epitope masking.
• Typical laboratory applications (research use only):
  • Light microscopy: General tissue/cell fixation prior to immunostaining (often followed by glycine/ammonium chloride quenching to reduce free aldehydes).
  • Electron microscopy (EM): Pre‑fixation with 1% glutaraldehyde enhances membrane and cytoskeletal preservation; commonly followed by osmium tetroxide postfixation (literature practice).
  • 3D cell culture and organoids: Structural stabilization prior to sectioning or clearing workflows (with appropriate permeabilization/clearing protocols).
• Practical tips:
  • Balance fixation time and temperature to preserve morphology while maintaining antigenicity; shorter times at RT or longer at 4 °C can mitigate epitope loss.
  • Rinse thoroughly after fixation (e.g., multiple buffer exchanges) and include quenching to minimize background fluorescence and residual reactivity.
  • For sensitive antigens, test postfix antigen retrieval or switch to PFA‑only fixation in pilot studies.
• Noteworthy constraints:
  • Primary amine–containing buffers during fixation decrease efficacy; use phosphate or HEPES buffers initially.
  • Over‑fixation can impair downstream enzymatic probes and nucleic acid accessibility—optimize empirically.

Reaction Conditions

The relevant “reaction” here is chemical fixation (crosslinking) of biological specimens. Conditions vary with sample type and downstream analysis. Values below are literature/general guidance, not item‑specific specifications.

• Temperature:
  • Room temperature (18–25 °C) for rapid fixation of cultured cells: 10–20 min typical.
  • 4 °C for delicate tissues or to reduce epitope masking: 30–120 min depending on thickness.
• Time vs. thickness:
  • Monolayer cells: 10–20 min.
  • Vibratome/cryosections (10–50 µm): 10–30 min.
  • Small tissue blocks for EM (≤1 mm): 30–90 min pre‑fix, followed by washes and osmium postfixation.
• Buffer/pH:
  • Neutral buffers (PBS/HEPES/phosphate or cacodylate) at pH 7.2–7.4 commonly used.
• Quenching (to reduce autofluorescence/free aldehydes):
  • 50–100 mM glycine or 50 mM ammonium chloride for 10–20 min; for high GA background, sodium borohydride (fresh, cold, caution: gas evolution) can be used on robust specimens.
• Permeabilization (post‑fix):
  • 0.1–0.3% Triton X‑100 or 0.05–0.1% saponin for 5–15 min depending on membrane sensitivity.
• Storage of fixed samples (short‑term):
  • After adequate washing, many specimens are stored in buffer at 4 °C for hours to days; prolonged storage in fixative is discouraged to limit over‑crosslinking.

Note: Optimize empirically for antigen preservation vs. morphology. Exact pH, buffer composition, and any additives for this SKU are Not specified for this item; refer to CoA/Spec Sheet.

Safety and Handling

• Safety status for this specific item: Full GHS classification, hazard statements, and pictograms are Not specified for this item; refer to the product SDS for authoritative information. The following are general hazards of aldehyde fixatives.
• General GHS profile of components (literature/general):
  • Formaldehyde: Flammable liquid/vapor (for concentrated solutions), acute toxicity (oral/dermal/inhalation), skin corrosion/irritation, serious eye damage/irritation, skin sensitizer, respiratory sensitizer, carcinogenicity (Cat. 1B). Strong lachrymator.
  • Glutaraldehyde: Acute toxicity (inhalation/dermal), skin corrosion/irritation, serious eye irritation, skin sensitizer, respiratory irritant; harmful/toxic to aquatic life.
• Primary routes of exposure: Inhalation of vapors/aerosols; skin and eye contact; accidental ingestion.
• PPE (good laboratory practice):
  • Chemical-resistant gloves (e.g., nitrile; check permeation data), lab coat, splash goggles or face shield.
  • Use in a certified chemical fume hood; avoid breathing vapors and prevent aerosol generation.
• Handling precautions:
  • Keep containers tightly closed; open only in ventilated enclosures.
  • Avoid contact with amines, strong oxidizers, strong bases/acids that can promote polymerization/condensation; avoid reducing agents.
  • Do not mix with bleach (hypochlorite) due to potential formation of toxic gases.
• First aid (overview; consult SDS):
  • Inhalation: Move to fresh air; seek medical attention. Skin: Remove contaminated clothing; wash with soap/water. Eyes: Rinse cautiously with water for several minutes; get medical attention. Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Spill/cleanup:
  • Contain and absorb with inert material; neutralize aldehydes with appropriate scavengers (e.g., ammonia/glycine solutions) if permitted by your institutional procedures; collect for disposal as hazardous waste.
• Fire: Use water fog, CO2, dry chemical; cool containers. Vapors may irritate responders.

Solvent Selection

This product is a ready‑to‑use aqueous mixed fixative, not a general-purpose solvent. Solvent selection per se is not typically applicable. However, compatibility with common biological buffers and media is critical.

• Polarity/miscibility (general):
  • Fully miscible with water; readily dilutable with standard isotonic buffers.
  • Compatible with phosphate‑buffered saline (PBS), HEPES, MOPS, and EM buffers (e.g., 0.1 M phosphate or cacodylate), provided pH/osmolarity are appropriately controlled.
• When to use as‑is vs. adjust:
  • Use as‑is for routine chemical fixation where 4% formaldehyde/1% glutaraldehyde is desired.
  • If osmotic balance is crucial (e.g., EM), ensure the diluent/buffer matches specimen osmolarity; verify whether this SKU is pre‑buffered (Not specified for this item; refer to CoA/Spec Sheet).
• Incompatibilities (general):
  • Do not mix with primary amine‑rich buffers (e.g., unbuffered Tris) during fixation; amines consume aldehydes and reduce effective crosslinking. Tris is suitable for post‑fix washes only after fixation is complete.
  • Avoid hypochlorite (bleach) mixtures and strong reducing agents.
• Alternatives/adjuncts:
  • For fluorescence imaging where autofluorescence is problematic, a 4% paraformaldehyde‑only fixative may be preferable; for ultrastructure, retain the glutaraldehyde component.
• Practical tip: Pre‑equilibrate fixative to the intended incubation temperature (RT or 4 °C) and pH to avoid osmotic or thermal shock artifacts.

Storage and Reconstitution

• Storage conditions (from Product Data): Store at 2–8 °C. Protect from extreme temperatures. Shipment on wet ice (from Product Data).
• Stability/shelf life after receipt: Not specified for this item; refer to CoA/Spec Sheet. As a general note for aldehyde fixatives, prolonged storage can lead to polymerization (formaldehyde) and self‑condensation (glutaraldehyde), potentially altering performance.
• Freeze–thaw guidance: Avoid freezing unless validated; freeze–thaw can induce phase changes/precipitates and alter effective concentration.
• Light/air precautions (general): Store tightly closed to minimize volatilization and oxidation; protect from strong light and contamination. Use clean, aldehyde‑compatible caps/septa.
• Handling after opening:
  • Record first‑open date; where possible, aliquot into airtight containers to limit headspace and repeated warming/cooling cycles.
  • Inspect visually before use; discard if turbidity, precipitate, or significant yellowing develops, unless validated as acceptable for your application.
• Reconstitution/dilution:
  • This product is supplied ready‑to‑use; no reconstitution required. If dilution or buffer adjustment is desired, use freshly prepared, pH‑controlled buffer matched for osmolarity and free of primary amines during fixation.
• Compatibility and container materials: Use glass or high‑quality HDPE/PP. Avoid reactive materials and natural rubber.

Always consult the product CoA and SDS for lot‑specific storage guidance and safety measures.

Structure and Identity

A ready-to-use, aqueous mixed aldehyde fixative containing paraformaldehyde-derived formaldehyde (4% w/v) and glutaraldehyde (1% w/v), optimized for rapid penetration and strong crosslinking in cell and tissue preservation.

• Item composition (from Product Name):
  • Formaldehyde (from depolymerized paraformaldehyde): 4% (w/v)
  • Glutaraldehyde: 1% (w/v)
  • Solvent/base matrix: Not specified for this item; refer to CoA/Spec Sheet.
• Mixture identity: No single molecular formula or SMILES applies; this is a defined aqueous mixture of two monofunctional/difunctional aldehydes.
• Functional groups (general chemistry):
  • Formaldehyde: one aldehyde (–CHO)
  • Glutaraldehyde: dialdehyde (OHC–(CH2)3–CHO)
• Structural description in words (literature/general):
  • Formaldehyde (monomer) is the simplest aldehyde; in water it exists largely as methanediol and short oligomers.
  • Glutaraldehyde is a five‑carbon dialdehyde that exists as a mixture of monomeric, hydrated, and oligomeric forms in water; these forms interconvert and contribute to crosslinking performance.
• Representative identifiers for the individual components (literature/general values, for reference only):
  • Formaldehyde: MF = CH2O; MW = 30.03 g/mol; SMILES: C=O; InChIKey: WFKWXMTUELFFGS-UHFFFAOYSA-N
  • Glutaraldehyde: MF = C5H8O2; MW = 100.12 g/mol; SMILES: O=CCCC=O (linear dialdehyde); InChIKey: DQGQKZPWVWRDNS-UHFFFAOYSA-N
• Stereochemistry: Not applicable (acyclic, achiral aldehydes).

Synthetic Utility

This SKU is a dilute aqueous fixative mixture, not typically employed as a reagent for synthetic organic chemistry. For completeness, the underlying aldehydes have the following reagent properties (literature/general), though users should procure neat or concentrated grades for synthesis:

• Formaldehyde (from paraformaldehyde):
  • Carbonyl electrophile for hydroxymethylation, Mannich reactions (with amines/enols), and as a C1 building block (e.g., Eschenmoser–Claisen variants). Often used as paraformaldehyde in aprotic media.
• Glutaraldehyde:
  • Bifunctional aldehyde useful for polymer crosslinking and as a spacer in biomolecule conjugation; can participate in aldol condensations and Schiff base formations.

Constraints for this item:

• Water content (aqueous matrix) and low concentrations (4%/1%) limit synthetic applicability and drive side reactions (hydrate/oligomer equilibria).
• Presence/absence of stabilizers, metal content, and UV cutoff are Not specified for this item; refer to CoA/Spec Sheet. These parameters are usually critical for synthetic photochemistry or catalysis and are unsuitable if unknown.

Recommendation: Use dedicated, specification‑controlled reagents (e.g., paraformaldehyde solid; glutaraldehyde 25% aq.) for synthesis. Reserve this mixed fixative for its intended role in sample preservation.

Target Specificity

Not applicable. This product is a general chemical fixative and does not possess biological target specificity, nor is it an antibody, probe, or enzyme. No antigen/epitope reactivity, clone, or isotype data apply.