Image-iT™ Fixative Solutions

Image-iT Fixative Solutions are ready-to-use fixative solutions provided in various formats to meet a variety of research needs. They are ideal for use with fluorescent proteins, such as GFP and RFP, and other small dye fluorophores. No more mixing of toxic fixative powders or dilution of concentrated solutions with non-optimal pH.  Just add these fixative solutions to cells/tissue and incubate for 10-15 minutes at room temperature. They are ideal for use with any antibody-based detection method, including immunocytochemistry (ICC), immunofluorescence (IF), and immunohistochemistry (IHC).

Features of Image-iT Fixative Solutions include:
• Ready-to-use
• High-purity, imaging-grade fixatives with minimum background signal artifacts
• Methanol-free
• Optimized for secondary antibody labeling, fixed-cell dye staining, and colorimetric IHC

4% Formaldehyde or Paraformaldehyde Fixative Solutions
4% formaldehyde and paraformaldehyde are the most commonly used fixatives for immunocytochemistry (ICC), immunofluorescence (IF), and immunohistochemistry (IHC).  Image-IT™ Paraformaldehyde 4% in 0.1M Phosphate Buffer pH 7.4 (Cat. No. I28800), is provided in ten vials pack with 10 mL each. Image-iT™ Formaldehyde 4% in PBS is provided in 20 mL size (Cat. No. FB002) or 5 x 20 mL size (Cat. No. R37814). Paraformaldehyde/formaldehyde 4% in 0.1 M phosphate buffer has ∼300 mOsm less osmolarity than in PBS, which helps maintain cellular morphology before the fixative has a chance to fix the inner cellular compartments. For paraformaldehyde/formaldehyde in PBS or phosphate buffer, just add the fixative to cells and incubate for 5-10 minutes at room temperature. Wash the cells in PBS after incubation and proceed to the blocking step.

Paraformaldehyde 3%, Glutaraldehyde 0.35% Fixative Solution
3% paraformaldehyde with 0.35% glutaraldehyde is a ready-to-use solution provided in methanol free, 0.1 M sodium cacodylate buffer. Yuan Qin, et al. (1) found that a mixture of paraformaldehyde and glutaraldehyde can preserve mitochondrial morphology better than either one alone.

Paraformaldehyde is small molecule that penetrates cells/tissue quickly, fixing proteins and other molecules in place. Glutaraldehyde is a bigger molecule with two reactive aldehyde groups that increase the potential for fixation and help create long chains of glutaraldehyde, generating a matrix. This ability helps preserve localization of proteins in complex environment such as mitochondria.

To fix cells/tissue, 5–10 minutes of incubation at room temperature is recommended. With glutaraldehyde, increased autofluorescence can be observed due to increased presence of reactive aldehyde groups. To help reduce this autofluorescence, approximately seven minutes of incubation with 0.1M glycine or freshly prepared 0.1% (w/v) sodium borohydride is recommended after fixation but before blocking.

Glyoxal 3% Fixative Solution
Glyoxal is a smaller molecule then formaldehyde or glutaraldehyde. It penetrates cells/tissue quickly, fixing the proteins, and preserving the cellular morphology better then formaldehyde (2). Glyoxal 3% Fixative Solution is a ready-to-use fixative solution provided in two separate bottles: Component A (92 mL) and Component B (8 mL), containing absolute Ethanol, Glyoxal Solution, Acetic Acid and water. Mix solution A and solution B in a empty vial, vortex the two solutions together and bring to a pH of 4.5 with the addition of 1M NaOH. Once mixed, this solution must be used immediately. To fix the cells/tissue, incubate cell/tissue at room temperature for approximately 10 minutes.

References
1: Qin Y, Jiang W, Li A, Gao M, Liu H, Gao Y, Tian X, Gong G. The Combination of Paraformaldehyde and Glutaraldehyde Is a Potential Fixative for Mitochondria. Biomolecules. 2021 May 10;11(5):711.
2: Richter KN, et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J. 2018 Jan 4;37(1):139-159.