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STUMol®

Molecular Biology Tissue Fixative

Cat#s2832

 

 

STUMol® Molecular Biology Tissue Fixative is a methanol-based non-aqueous tissue fixative blended to meet the needs of plant and animal experimental biologists and molecular pathologists engaged in genomic and proteomic study of tissue.

 

STUMol® Molecular Biology Tissue Fixative is a preferred fixative for:

  • Genomic studies:  Both DNA and RNA can be recovered in superb quantity, and of excellent integrity, such that gene expression studies may be performed successfully.
  • Proteomic studies: All forms of proteins can be recovered unaltered by fixation; no need to perform heat/enzymatic recovery methods to free cross-linked proteins. Additionally, there is no need to pretreat tissue for immunohistochemical staining, and antibodies may be used at lower dilution, and at shorter incubation times.
  • Examination by light microscopy:  Tissues are fixed thoroughly without alteration of architecture or distortion of critical intracellular features.
  • Laser Capture Microdissection:  STUMol® Molecular Biology Tissue Fixative was developed with those who perform LCM in mind; effectively fix tissue such that cells of interest can be harvested which contain unaltered nucleic acids and undistorted proteins.

 

Tissues fixed in STUMol® Molecular Biology Tissue Fixative exhibit architecture and cellular morphology similar to that seen in formaldehyde-fixed tissue stained with both routine and special stains, to include immunostaining.

 

Some Background

 

Formaldehyde, as 10% formalin (40% formaldehyde stabilized with methanol and diluted with water to approximately 4% active strength,) is essentially the gold standard for preserving tissue for histologic study. On contact with water formaldehyde forms methylene glycol, which quickly penetrates tissue. Formaldehyde penetrates tissue as methylene glycol, but fixes tissue slowly as carbonyl formaldehyde 1.  Terminal amino groups are bonded to form methylene (-CH2-) bridges between adjacent proteins, referred to as cross-links. Cross-links insolubilize proteins, thus initiating their preservation. Formalin is excellent in preserving the morphology of tissue and intracellular features, but is not an efficient preservative of tissue macromolecules.

 

Reaction of formaldehyde with tissue DNA does not result in significant recovery of this macromolecule. Formaldehyde fixation at room temperature results in poor preservation of high molecular weight DNA. Up to 30% of nucleic acids may be lost during fixation.2  Formaldehyde-induced modifications of bases in nucleic acids (adducts) such as methylol (hydroxymethyl) and methylene bridge crosslinks block or reduce the base pairing necessary for molecular analysis by hybridization techniques. Formaldehyde is also responsible for cross-links to other macromolecules that reduce the yield of RNA.6   In DNA non-reproducible sequence alterations, caused by fixative-induced cross-links of cytosine nucleotides, form on either strand. The cross-linking of cytosine nucleotides by formalin is suspected to result in failure of Taq-DNA polymerase in PCR to recognize cytosine, and incorporates an adenine in place of a guanosine; an artificial C-T or G-A mutation is created. Up to 1 mutation artifact per 500 bases has been reported.3

 

 Formaldehyde is not a good choice as a fixative to preserve RNA quality and quantity. In one study, formalin fixation resulted in a loss of 80% of RNA information content.4  RNA is extensively degraded by formalin fixation to fragments averaging 200 nucleotides.Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. “This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens.”Reproducible RT-PCR on formal-fixed paraffin-embedded extracted RNA is limited to amplicons of fewer than 300 bases.20

 

 

Formalin-induced cross-linking prevents unimpeded examination of macromolecules. “Formalin fixation results in profound changes in the conformation of macromolecules which could make the recognition of proteins by antibodies impossible, or at best difficult.”8  Formaldehyde-induced cross-links change the three-dimensional (tertiary and quaternary) structure of proteins, while primary and secondary proteins are not badly affected.In order to expose epitopes to recognition, retrieval methods using heat, pressure and sometimes enzymes must be used. Once exposed, many antigens can be visualized using specific antibodies with appropriate indicators

 

Alcohol Fixatives

 

Alcohol fixatives evolved as alternatives to formaldehyde, and in many cases, offer a marked improvement in fixation utility. Ethanol and methanol are considered coagulants that denature proteins by replacing water in the tissue environment; this disrupts hydrophobic and hydrogen bonding in proteins, exposing internal hydrophobic groups of the proteins, thus altering their structure and solubility in water.10  Essentially, the proteins collapse. Tissue fixation with ethanol commences at 50% to 60% concentration, while with methanol concentration must be at >80%.11

There is a consequence to using concentrated ethanol and methanol as primary fixatives for routine applications, as tissue shrinkage and hardness or brittle tissues occur. It has been long known that the addition of a weak acid, such as acetic acid, counters the dehydrating effect of methanol and ethanol fixatives.

 

70% ethanol has been used in studies for the recovery and assessment of macromolecules. Gillespie et al 12 evaluated 70% ethanol as a tissue fixative. For preserving tissues such that a subjective clinical diagnosis could be made using light microscopy, there were no reported problems. The appearance of tissue architecture and nuclear detail of cells was comparable to that seen in formaldehyde-fixed tissue. DNA recovered from ethanol-fixed tissue consistently amplified much stronger than that from formaldehyde-fixed tissue. RNA recovered from ethanol-fixed tissue was of reduced quality when compared from RNA derived from fresh-frozen tissue. Intact 18S and 28S ribosomal RNA bands were clearly detected in pherograms of RNA derived from fresh-frozen tissue, indicative of high-integrity RNA. By contrast, pherograms of ethanol-fixed tissue RNA failed to produce 18S and 28S ribosomal RNA bands.  Nonetheless, isolated RNA was sufficient to perform a number of molecular techniques such as gene-specific RT-PCR.  In the evaluation of

protein quality and quantity derived from ethanol-fixed tissue using one-dimensional gel electrophoresis, results were similar to that from snap-frozen tissue, but superior to recovery from formaldehyde-fixed tissue.  Su et al.13  found 70% ethanol to be a superior fixative to formaldehyde for mRNA preservation, with yields 70% of that from fresh-frozen tissue. Only negligible quality RNA was recovered from formaldehyde-fixed tissue. After laser capture dissection recovery of cells from formalin-fixed tissue, mRNA was not recovered, but from ethanol-fixed tissue, sufficient mRNA was recovered for RT-PCR and cDNA microarray analysis.

 

While the use of fresh tissue is the ultimate source for recovery of macromolecules, fresh-frozen tissue is also a rich source of DNA, RNA and proteins. The price paid for using frozen tissue is the reduced morphological quality for examination by light microscopy.  Absolute methanol and ethanol are known to be excellent fixatives for preserving both high molecular weight DNA and RNA.14  Tissue shrinkage is a major concern when using absolute ethanol and methanol as fixatives.

 

Some Alcohol Variants

 

Some alcohol-based fixative blends have been developed for examining tissue for molecular profiling. Carnoy’s fixative is a blend of ethanol, chloroform and acetic acid. A modification of Carnoy’s fixative is Methacarn fixative, which is similar to Carnoy’s fixative, but contains methanol substituted in place of ethanol. Park et al. found the ability of Carnoy’s and methacarn fixatives to preserve cell morphology well enough that 60% of prepared slides could be read satisfactorily by light microscopy. Additionally, tissue fixation with methacarn gave 3.5 times greater expression of GAPDH than when using formalin for gene expression studies after laser capture microdissection.15  But a note of caution: Carnoy’s  fixative and methacarn fixative must be prepared immediately prior to use, and chloroform is highly flammable. Consistent content quality is an important consideration.

 

In time methacarn fixative became modified, sometimes by the substitution of chloroform by other ingredients, such as 1,1,1-trichlororthne16 , or simply the elimination of chloroform from the formulation without substitution. STUMol ® Molecular Biology Tissue Fixative is a modified methacarn type fixative with ingredients added for membrane protection and permeation enhancement.

 

Cox et al.18 detailed their assessment of modified methacarn tissue fixative. Morphological quality of rat liver sections fixed in modified methacarn, 70% ethanol and 10% neutral buffered formalin were compared and ranked on appearance of nuclear detail, cytoplasmic detail and cell membrane detail after standard fixation and processing protocols were followed. Modified methacarn fixed tissues were found to be superior to those fixed in 70% ethanol and in 10% neutral buffered formalin. In terms of RNA quality and quantity, as measured using UV spectrophotometry from tissue isolates, the highest yields were obtained from tissues which were snap frozen. Tissue fixed in formalin had a yield which did not exceed 2µg. Tissue fixed in 70% ethanol yielded in the low 40µg range, while those fixed in modified methacarn yielded between 35µg and 40µg.  Quality of RNA from tissue fixed in 70% ethanol, 10% neutral buffered formalin, and modified Methacarn were compared. RNA from snap-frozen liver served as the control. Frozen tissue lost 5% of the information contained in the snap-frozen control specimen, liver fixed in 70% ethanol lost roughly 25% content, and 80% content was lost in liver fixed in 10% neutral buffered formalin. Impressively, liver fixed in modified methacarn lost only about 10% of RNA information content. “Modified methacarn was the best fixative for the combined analysis of morphology, RNA quality, and efficiency of amplification from LCM samples.”18   Mitchell et al. used modified methacarn to fix tissues for immunohistochemical staining. Protein-precipitating agents fixed tissues producing superior immunohistochemical staining when compared with cross-linking fixatives. Tissues fixed in non-crosslinking alcohol-based fixatives can successfully be immunohistochemically stained following the usual neutral buffered formalin protocols. Omission of pepsin treatment seems to be important to retain proper morphology of immunostained tissues preserved in alcohol-based fixatives.19

 

Formaldehyde is an excellent tissue fixative, but not for molecular studies. Ethanol is an improvement over formaldehyde, but sometimes falls short in utility for molecular studies. Goldsworthy et al. stated: “optimal fixation must provide acceptable morphology, allow laser capture of selected cells, and preserve the integrity of mRNA”. Further, “Optimal fixation protocols for laser capture microdissection analysis will facilitate the examination of gene expressions in selected cell populations, accelerating investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis.17  This aptly describes the utility of STUMol® Molecular Biology Tissue Fixative, which was developed for use in LCM.

 

 Notes for using STUMol® Molecular Biology Tissue Fixative

 

Cut tissue to not more than 3mm x 3mm x 3mm .  Preferred size is 1.5mm x 1.5mm x 1.5mm

Immerse cut tissue in 20 volumes of fixative to tissue mass.

Fix tissue for at least 24 hours at room temperature. Recommended total fixation time is 48 to 72 hours.

Tissue culture cells should be gently suspended in about 1 ml. of fixative.

Cytological specimens should be suspended in about 1 ml of fixative.

Punch biopsies should be placed in closed vials containing between 1 and 2 ml of fixative.

 

Tissue Processing Using Automated Machines

 

95%  ethanol                            40 degrees C.                    45 minutes

95%  ethanol                            40 degrees C.                    45 minutes

100% ethanol                           40 degrees C.                    45 minutes

100% ethanol                           40 degrees C.                    45 minutes

100% ethanol                           40 degrees C.                    45 minutes

Paraffin wax                             58 degrees C.                    30 minutes

Paraffin wax                             58 degrees C.                    30 minutes

Paraffin wax                             58 degrees C.                    30 minutes

Paraffin wax                             58 degrees C.                    30 minutes

Embed into tissue block with molten paraffin

 

Microwave fixation, using STUMol® Molecular Biology Tissue Fixative, may also be used, following the same protocol for time, temperature and power settings as with microwave fixation of tissue using 10% formalin

 

Suggested Microwave Processing Protocol

 

100% ethanol           55 degrees C.              15 minutes        100% power

100% ethanol           65 degrees C.              15 minutes        100% power

Isopropanol              65 degrees C.              15 minutes        100% power

Isopropanol              65 degrees C.              15 minutes        100% power

Paraffin wax             70 degrees C.              20 minutes        100% power

Embed into tissue block with molten paraffin

 

Microwave processing does not necessarily improve RNA preservation. “We found that the fixative is the most important factor in the preservation of RNA quality, and while microwave protocols can modestly improve morphology, they have no significant impact on RNA preservation. Modified methacarn was the best fixative for the combined analysis of morphology, RNA quality and efficiency of amplification from LCM samples.” 18

 

Hematoxylin and Eosin Staining

 

Using a microtome, cut sections to 4µm to 5µm thick sections and place onto glass slides. Poly-l-lysine coated or silane treated slides are recommended. All slides must be RNAase free.

Dewax in two consecutive baths of xylene for 5 minutes. Place slides in baths of 100% ethanol for 10 seconds, 95% ethanol for 10 seconds, then 70% ethanol for 10 seconds.

 

Stain with hematoxylin for 45 seconds

Wash with 3 dips in normal saline

Counter-stain with eosin for 45 seconds to 2 minutes. (adjust to preference)

Wash with normal saline

Dehydrate with 70% methanol for 10 to 15 seconds, 95% methanol for 10 to 15 seconds, then finally with 100% methanol for 20 to 30 seconds

 

Immunohistochemical Stains

 

There is no need to perform enzymatic or heat-induced epitope retrieval procedures prior to staining, since STUMol® Molecular Biology Tissue Fixative is a non-crosslinking fixative.

 

Follow manufacturer’s guidelines for staining and counterstaining. Antibodies may be used at higher dilutions-determine empirically.

 

Commercially available products may be used with cells and tissues fixed in STUMol® Molecular Biology Tissue Fixative for the isolation of DNA, RNA and proteins. TRIzol Reagent  (Life Technologies) and TRI Reagent  (Sigma-Aldrich) are excellent products for these procedures

 

STUMol® is a registered trademark of Advanced Fixatives Technology, LLC  Timonium, Maryland  USA

 

 

References

 

  1. 1.       Srinivasan, M et al.

Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids

Am J Pathol. 2002; 161(6):  1961-1971

 

  1. 2.       Cross, SS et al.

Does delay in fixation affect the number of mitotic figures in processed tissue?

J Clin Pathol.  1990;  43: 597-599

 

  1. 3.       Williams, C et al.

A high frequency of sequence alterations is due to formalin fixation of archival specimens

Am J Pathol. 1999; 155: 1467-1471

 

  1. 4.       Cox, M et al.

Investigating fixative-induced changes in RNA quality and utility by microarray analysis

Exp Mol Pathol.  2008; 84(2):  156-72

 

  1. 5.       Krafft, AE et al.

Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue: The Armed Forces Institute of Pathology experience and literature review

Mol Diagn.  1997; 2: 2217-230

 

  1. 6.       Feldman, MY

Reactions of nucleic acids and nucleoproteins with formaldehyde

Prog Nucleic Acid Res Mol Biol  1973;  13:  1-49

 

  1. 7.       Evers, DL et al.

The Effect of Formaldehyde Fixation on RNA

J Mol Diagn.  2011; 13(3):  282-288

 

  1. 8.        Montero, C et al

The antigen-antibody reaction in immunochemistry

J Histochem Cytochem  2003; 51(1): 1-4

 

  1. 9.        Ramos-Vara, JA

Technical Aspects of Immunochemistry

Veterinary Pathology  2005;  42(4):  405-426

 

  1. 10.    Fixation and Fixatives

Rolls, Geoffrey

Leica Biosystems

 

  1. 11.    Fixation and Fixatives (3)

Rolls, Geoffrey

Leica Biosystems

 

  1. 12.    Gillespie, JW et al.

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Am J Pathol.  2002; 160(2):  449-457

 

  1. 13.    Su, JMF et al.

Comparison of Ethanol Versus Formalin Fixation on Preservation of Histology and RNA in Laser Capture Microdissected Brain Tissues

Brain Pathology  2004; 14(2):  175-182

 

 

    14.   Srinivasan, M et al.

Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids

Am J Pathol.  2002;  161(6):  1961-1971

  

 

  1. 15.    Park, IS et al.

Impact of fixatives on preservation of RNA in paraffin-embedded and laser capture microdissected human tissues

Basic and Applied Pathology  2008; 1:  72-76

 

  1. Mitchell, D et al.

Improved Immunohistochemical Localization of Tissue Antigens Using Modified Methacarn Fixation

J Histochem Cytochem 1985; 33(5):  491-495

 

  1. 17.    Goldsworthy, SM et al.

Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue

Mol Carcinog.  1999; 25(2):  86-91

 

  1. 18.    Cox, M et al.

Assessment of fixatives, fixation and tissue processing on morphology and RNA integrity

Experimental and Molecular Pathology  2006; 80:  183-191

 

  1. Mitchell, D et al.

Improved Immunohistochemical Localization of Tissue Antigens Using Modified Methacarn Fixation

J Histochem Cytochem 1985;  33(5):  491-495

 

  1. 20.    Chung, JY et al.

Optimization of recovery of RNA from formalin-fixed paraffin-embedded tissue

Diagn. Mol Pathol. 2006; 15: 229-236

 

On the following page there are three bioanalyzer generated pherograms which compare isolates from mouse liver tissues fixed in STUMol® Molecular Biology Tissue Fixative, with frozen mouse liver, as the control.

Graph 1 depicts the results of the control. RNA concentration is 4284pg/µl. RIN is 9.2

18S and 28S ribosomal bands clearly evident.

 

Graph 2 depicts the results of tissue fixed in STUMol® Molecular Biology Tissue Fixative, but not processed. RNA concentration is 275ng/µl. RIN is 7.1   18S and 28S bands clearly visible.

 

Graph 3 depicts the results of tissue fixed in STUMol® Molecular Biology Tissue Fixative, then processed to paraffin.  RNA concentration is 262ng/µl. RIN is 5.2  Note 18S and 28S bands.

 

RNA from tissue fixed in STUMol® Molecular Biology Tissue Fixative, but not taken to paraffin blocks is an excellent source of RNA suitable for downstream applications, including microarray.

Tissue fixed in STUMol® Molecular Biology Tissue Fixative then paraffin-embedded utilizing laser capture microdissection is suitable for amplification and further study.

 

Electropherograms credit:  PHL of Leidos Biomedical Inc.

Lawrence Sternberg, Ph.D (Lab Head), Andrew Warner, MS, Tamara Morgan, HT(ASCP) and Yelena Golubeva, Ph.D