The influence of fixatives and other variations in tissue processing on nuclear morphometric features

The influence on the area and numerical density of nuclei was investigated in 5-mm-thick slices of guinea pig liver for different fixatives and variations in tissue processing: delay in fixation, air drying, degree of acidity of 10% formalin (= 4% formaldehyde), Bouin and mercury-formalin fixatives, acetone and ethanol dehydration and understretching and overstretching of the paraffin-embedded sections. Air drying (either forced or as a result of delayed fixation), the type of fixative and the degree of acidity affected the nuclear area. Regarding the latter, nuclear area was approximately 25% lower for pH less than or equal to 3 as compared with pH greater than 5. In comparison with the standard tissue processing used, the nuclear density was higher after all of the variations studied (air drying, acetone dehydration and fixation). These findings indicate that nuclear area, in contrast to other tissue components, is relatively insensitive to variations in tissue processing. However, it is essential to regularly measure the pH of the fixative: deviations from pH = 7 should be carefully avoided in order to keep nuclear area variations as a result of tissue processing within acceptable limits.     

Influence of initial fixation protocol on the appearance of primary cilia on the rabbit corneal endothelial cell apical surface as assessed by scanning electron microscopy

The objective was to examine primary cilia at the apical surface of corneal endothelial cells after using different fixatives. Female albino rabbits (2 kg) were euthanised at 15:00 h and the corneas fixed immediately (usually with an isotonic 2% glutaraldehyde-cacodylate fixative) either after dissection, by application fixative at 4 degrees C, by immersion of the eyeball in fixative at room temperature (RT), or by application of an isotonic or a hypertonic (Karnovsky-type) fixative at RT. Images at 2000x were taken from the central corneal region, and number and length of primary cilia assessed. The length was the same regardless of method (overall average of 1.67+/-0.70 microm), but the incidence of primary cilia was hypertonic fixative (87% of cells) >cold drop fixation (71%), >whole globe immersion (68%) >dissect then fix methods (67%) >RT drop fixation (34%). The first four methods however yielded cells with unacceptable artefacts (especially distortion). More details should be provided of the primary fixation method used.     

Vimentin-typing in diagnostic surgical pathology: a comparative study using four antibodies after different fixations

Vimentin-typing was carried out on various normal and neoplastic tissues using four anti-vimentin antibodies in order to evaluate the effect of different fixation treatments on tissue reactivity in comparison to the results obtained on frozen sections. All antisera were reactive on frozen material; on paraffin embedded material staining of tissues depended on the type of fixation method applied (formalin, methacarn or absolute alcohol) and each antibody behaved differently in relation to the fixative used. Only mesenchymal normal structures were revealed on frozen material whilst on paraffin embedded material three of the four antibodies reacted also with non-mesenchymal normal structures (epithelia, central and peripheral nervous system cells). All four antibodies decorated, regardless of treatment, neoplastic cells of mesenchymal and non-mesenchymal derivation, but not germ cells or germ cell tumors. The reactivity of vimentin to its specific antibodies depends on the fixative used: therefore, in routine pathology more than one antiserum should be available for testing. Furthermore, given the variety of non-mesenchymal structures stained by the anti-vimentin antibodies, the differential diagnosis of undifferentiated tumors must not be based on vimentin positivity alone. The expression of vimentin by non-mesenchymal neoplastic cells seems to parallel that of normal tissues during embryogenesis; therefore, this intermediate filament appears to be not only a marker of mesenchymal cells but also of many immature elements.     

Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium

The purpose of this study was to examine the impact of a low osmolality glutaraldehyde fixative and a high osmolality glutaraldehyde-formaldehyde fixative on the structural organization of a tissue that could be exposed to low and high osmolality environments. The corneas of freshwater trout were prepared for transmission and scanning electron microscopy using either a fixative of 2% glutaraldehyde in 60 mM cacodylate buffer (pH 7.8, 260 mOsm/l) or a fixative prepared by adding 2.5% glutaraldehyde to a solution of 1% formaldehyde and buffering the solution with 0.1 M cacodylate (pH 7.6, 850 mOsm/l; Karnovsky-type fixative). The corneal epithelial cell layer thickness was greater after glutaraldehyde compared to glutaraldehyde-formaldehyde fixation (67 vs 55 mum), as was the thickness of the superficial cells (5.1 vs 3.4 mum) and basal cells (43 vs 38 mum). The intermediate (wing) cells of the epithelium were, however, less thick after glutaraldehyde fixation (15 vs 18 mum). The width of the squamous, intermediate and basal cells was greater following glutaraldehyde fixation with the effect being greatest in the superficial layers and insignificant at the level of the basal cells. The results show that chemical fixatives with extremes of osmolality cannot only produce different cell sizes in a tissue but also determine the overall organization of the cells in a positional-dependent fashion.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes. A morphometric study of so-called "dark cells" and their putative role in epidermal carcinogenesis

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered "clear cells" dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reproducibility of nuclear morphometric measurements in benign human epithelial cells. Simulation of variations in routine tissue processing.

The value of nuclear morphometric measurements in diagnostic pathology is determined largely by the reproducibility of the measurements. Although a variety of factors have been shown to affect tissues during processing, the regulation of fixative type and the avoidance of air drying in particular have been shown to avoid significant variations in nuclear measurements. The current study simulated routine tissue processing in order to identify any factors that may introduce variability of nuclear morphometric values in day-to-day processing if air drying is avoided and fixative type and pH are regulated. Samples of benign endometrium were collected from three uteri, fixed in phosphate-buffered formalin (PBF) from 2 hours to 15 days and dehydrated in an automated tissue processor on four occasions. In addition, tissue from one case was cut at 4, 6 and 8 microns, simulating the potential variations in section thickness that may occur during routine processing. Mean nuclear areas and shape factors of epithelial cells were then determined using computed planimetry. By analysis of variance, no significant differences were found in nuclear morphometric values in relation to time of fixation, dehydration runs or tissue section thickness; coefficients of variation for all variables were less than 7%. This study suggested that routinely processed tissues are adequate for morphometric analysis, including retrospective analysis, provided that tissues are fixed in a pH-regulated fixative such as PBF and air drying is avoided.     

Enhanced preservation of endosecretory granules in PP-cells of the canine pancreas

Standard fixation techniques commonly used for light and electron microscopic studies have resulted in reported differences in the ultrastructural appearance of endosecretory granules of the pancreatic polypeptide (PP) cell. To clarify these differences, canine pancreatic tissues of intact and cultured pseudoislets were studied using a variety of ingredients, additives and fixatives in an effort to better preserve the endosecretory granules of PP cells. Results show that preservation of PP granules is enhanced by addition in zinc chloride (0.5%) to a glutaraldehyde-paraformaldehyde fixative in 0.1 M cacodylate buffer, followed by osmium tetroxide fixation. This fixative is recommended for all light and electron microscopic studies of the pancreatic polypeptide cell.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding.

1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

A comparison of several fixative solutions for histochemical studies on human gastric mucosa

The effects of different fixative solutions on the staining of polyanions and Paneth cell granules and on alkaline phosphatase activity were evaluated in surgical specimens of human gastric mucosa with areas of intestinal metaplasia, which were dehydrated and embedded with routine procedures. Alcohol-formol proved to be particularly advisable for studies on the epithelial mucins, buffered formol with cetylpyridinium chloride for the connective tissue polyanions and the fluid of Mota et al. (1956) for the mast cells. In areas of complete intestinal metaplasia, the Paneth cell granules were destroyed by acidic fixative mixtures and 95% ethanol; in the same areas, alkaline phosphatase activity was well demonstrated after fixation with formol, alcohol-formol, or 95% ethanol.     

Preservation of fine Structures in Yeast by Fixation in a Dimethyl Sulfoxide-Acrolein-Glutaraldehyde Solution

The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl₂, the yeast cells were postfixed in 3% OsO₄ at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure     

Lysozyme antigenicity and tissue fixation.

The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues aqueous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Improved demonstration of exocytotic profiles in glomus cells of rat carotid body after perfusion with glutaraldehyde fixative containing a high concentration of potassium

Extensive secretion by exocytosis was demonstrated in the glomus (type I) cells of the adult rat after perfusion of carotid bodies with a potassium-rich (high K) glutaraldehyde fixative. Similar secretory profiles were very rare with a glutaraldehyde fixative containing a low concentration of potassium (low K). The increase in the incidence of exocytotic profiles in glomus cells with the high K fixative was highly significant, whereas no statistical difference could be observed in the incidence of coated pits with the different fixatives. Exocytotic profiles were characterized by the following features: (1) they predominated in non-synaptic regions, but were occasionally observed near synapses between two glomus cells; they were not observed near synapses between glomus cells and nerve terminals; (2) extruded electron-dense material associated with coating of the cell membrane was frequent; (3) different stages of dissolution of the extruded granule material was evident. The possible role of exocytosis as a mode of secretion in the glomus cells and the characteristics of the new high K-glutaraldehyde fixative are discussed.     

A selective stain for mitotic figures, particularly in the developing brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

Nuclear volume in type I gastric intestinal metaplasia

OBJECTIVE: To retrospectively compare nuclear size of epithelial cells in type I intestinal metaplasia (IM) from various pathologic lesions of gastric mucosa. STUDY DESIGN: Endoscopic mucosal biopsies with type I IM from intestinal type gastric carcinoma (n = 25), chronic gastritis (n = 40) and benign ulcer (n = 32) cases were analyzed. After standard fixation, embedding, sectioning, routine hematoxylin and eosin and alcian blue--periodic acid--Schiff reaction (pH 1.0 and 2.5) staining were used for identification of IM. The mean point sampled nuclear intercept was estimated by the original test system and 100x objective at a total magnification of 1,200x. To obtain the mean nuclear volume, the cubed nuclear intercept was multiplied by pi/3. In each case 100 epithelial cell nuclei were analyzed. RESULTS: In type I IM in gastric carcinoma cases there was significantly greater nuclear volume (118.34 +/- 10.32 micron 3) than in type I IM in other pathologic states of gastric mucosa (77.72 +/- 8.58 micron 3). CONCLUSION: The karyometric findings of the present study suggest a difference between type I IM found in benign pathologic states and type I IM found in gastric mucosa surrounding carcinoma, despite morphologic and histochemical similarities. Nuclear volume may be used in early detection of precancerous states of gastric mucosa.     

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.