Immunohistochemical procedures for the demonstration of peptide- and tyrosine hydroxylase-containing nerve fibers in cryostat sections of unfixed rapidly frozen tissue stored for long periods of time

Summary Traditional protocols for the immunohistochemical localization of peptides and tyrosine hydroxylase (TH) in nerve fibers in cryostat sections require the tissue to be thoroughly fixed and rinsed and to be processed for the cryostat sectioning and the immunohistochemical staining more or less directly after freezing. In the present study it was tested whether also unfixed, rapidly frozen tissue, conforming to guinea pig and bovine heart specimens, can be used for the visualization of neuropeptides [neuropeptide Y (NPY) and substance P (S P)] and TH in cryostat sections. The following observations were made: 1) NPY-immunoreactive (IR) and S P-IR nerve fibers could be clearly identified in both fixed and unfixed sections of this type of tissue. 2) TH-IR nerve fibers could be detected in unfixed tissue if the sections were post-fixed with aldehydes by the use of a two-step fixation process related to a sudden change of pH. However, the outlines of the nerve fibers were sometimes diffuse. 3) Storage of unfixed tissue for periods of up to 2.5 yeart at –80° C did not lead to a decrease in immunoreactivity. 4) Somewhat higher concentrations of primary antibodies had to be used for sections of unfixed tissue than for sections of fixed tissue when the FITC method was used. This waste of antibodies was partly overcome by use of the biotin-streptavidin method. The glyoxylic acid induced catecholamine(CA)-fluorescence method for demonstration of sympathetic nerve fibers was also applied and was found to give optimal results after storage of tissue for up to 2.5 years. The study shows that the use of unfixed rapidly frozen tissue represents a fast and realistic method for the demonstration of neuropeptide immunoreactivity, that it to some extent can be used for the visualization of TH-containing nerve fibers and that it is a suitable method to maintain longterm neuropeptide and TH immunoreactivity as well as long-term CA-fluorescence reaction.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections

We describe a novel immunohistochemical technique which permits the detection of specific binding of human monoclonal antibodies (MAb) to cryostat sections of human tissues. The technique overcomes the problem of background staining caused by the presence of endogenous immunoglobulins in tissue sections. This is achieved by the formation of a molecular complex of the primary antibody (a human MAb), horseradish peroxidase-conjugated goat anti-human immunoglobulin, and normal human serum. This complex is then incubated with cryostat sections of human tissue, and binding of the complex is demonstrated using diaminobenzidine/hydrogen peroxide. The method is suitable for immunohistochemical screening of small samples of tissue culture supernatant for the presence of human MAb of potential interest, and for determining the pattern of binding of such MAb to a wide range of normal and pathological human tissues.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Differential immunohistochemical resolution of the human mononuclear phagocyte system

Four new monoclonal antibodies, termed Ki-M1, Ki-M2, Ki-M3, and Ki-M4, were developed for distinguishing macrophage subgroups. Purified lysosomes of cells of stimulated U-937 cell line were used as immunogen. Specificity control was performed by staining unfixed cryostat sections of fresh human tissue with an immunohistochemical method, which allowed reliable recognition of reactive structures. Ki-M1 reacted with macrophages of lymphoid tissue, lung, and serous cavities. Ki-M2 recognized Kupffer cells and splenic macrophages. Both monoclonal antibodies reacted with interdigitating reticulum cells and Langerhans cells, which are thought to be accessory cells of the T-cell immune response. Accessory cells of the B-cell immune response, on the other hand, showed reactivity with Ki-M3 and Ki-M4. Thus, analogous to T lymphocytes, the human mononuclear phagocyte system (MPS) can be subdivided into different subgroups with the aid of appropriate monoclonal antibodies.     

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section

Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Techniques for studying adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl2 (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl2 (2.5%). When procedures described above for immature adipose depots are combined with esterase staining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Summary The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Specific distribution pattern of nerve fibers containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON) in the pineal gland of the chinchilla (Chinchilla laniger)--an immunohistochemical study

The sympathetic nerve fibers originating from the superior cervical ganglia and supplying the pineal gland play the most important role in the control of the pineal activity in mammals. NPY and CPON are also present in the majority of the pinealopetal sympathetic neurons. In this study, immunohistochemical techniques were used to demonstrate the existence and coexistence of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) as well as NPY and CPON in the nerve fibers supplying the chinchilla pineal gland. Ten two-year-old female chinchillas housed in natural light conditions were used in the study. The pineals were fixed by perfusion. ABC immunohistochemical technique and immunofluorescence labelling method were employed. TH-immunoreactive (TH-IR) varicose nerve fibers were observed in the pineal gland as well as in the posterior commissural area. Within the chinchilla pineal gland, TH-IR nerve fibers were located in the capsule and connective tissue septa. Numerous varicose TH-IR branches penetrated into the parenchyma and formed a network showing the highest density in the proximal region of the gland. In the central and distal parts of the pineal parenchyma, a subtle network, composed of thin varicose nerve branches, was observed. Double immunostaining revealed that the majority of TH-IR nerve fibers was positive for DbetaH or NPY. TH- and DbetaH-positive neuron-like cells were observed in the proximal region of the gland. The pattern of pineal innervation immunoreactive to CPON was similar to the innervation containing NPY, TH and DbetaH. The chinchilla intrapineal innervation containing TH, DbetaH, NPY and CPON is characterized by the higher density in the proximal part of the gland than in the middle and distal ones. The specific feature of the chinchilla pineal is also the presence of single TH/DbetaH-immunoreactive neuron-like cells in the proximal part of the gland.     

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues.     

A novel technique for immunohistoperoxidase staining of unfixed whole joints of small animals

Summary A method has been developed to cut unfixed and undecalcified sections of rat paws from animals with adjuvant arthritis and to stain them by a biotin-avidin immunoperoxidase technique. Good tissue integrity and morphology throughout the immunohistochemical procedure were retained if the sections were first mounted on transparent sellotape. The method is illustrated with two monoclonal antibodies (mAb) and is generally applicable with any mAb or polyclonal antibody and with joints from other small animals. For rats with adjuvant arthritis it was found important to block endogenous peroxidase before immunostaining. Complete inhibition of this enzyme without loss of antigenicity was best achieved after application of the mAb and biotinylated anti-IgG conjugate to the unfixed tissue sections.     

A differential stain for neuronal nucleoli in unfixed cryostat sections.

The Klüver-Barrera procedure, using luxol fast blue and cresyl violet for a combined nissl and myelin stain, was adapted to unfixed cryostat sections. Neuronal nucleoli appeared as distinct dark blue structures. The color contrast between violet Nissl substance and the nucleoli facilitated their recognition in human and in rat central nervous systems. This modified staining procedure enabled us to combine a counting of nerve cells with a histochemical investigation by applying each technique to a different set of sections cut from the same block of unfixed, frozen brain tissue.     

Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies

Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at pH 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement.     

Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology

Summary Three enzymes in the catecholamine synthesis—dopa decarboxylase (DDC), dopamine--oxidase (DBH) and phenylethanolamine-N-methyltransferase (PNMT)—have been isolated. Subsequently antibodies to these enzymes were prepared and used in immunohistochemical studies mainly with the aim to elucidate methodological problems. The indirect immunofluorescent technique was used throughout the study.It was found that cryostat sectioning followed by fixation in acetone or alcohol, a standard procedure in immunohistochemistry, was successful only with antibodies to the granule bound enzyme DBH. With antibodies to DDC and PNMT, two cytoplasmic enzymes, on the other hand, the results were hampered by diffusion artefacts. These drawbacks could be prevented by a brief aldehyde fixation, preferably by perfusion before cryostat sectioning. The best results were obtained with formalin followed by hydroxyadipaldehyde and acrolein. However, after glutaraldehyde fixation no specific fluorescence at all was observed.Freezing of fresh adrenals, followed by freeze-drying, treatment with formaldehyde vapours and paraffin embedding was tested but consistent results were only obtained with antibodies to PNMT.A new instrument, the Vibratome^®, which allows sectioning of unembedded fixed or unfixed tissue, was used and successful results were obtained with all three antibodies. Furthermore, the possibility with this instrument to combine the immunohistochemical technique e.g. with the formaldehyde fluorescence method for visualization of monoamines is demonstrated. It is emphasized that the Vibratome ^® technique may be a valuable tool for immunohistochemical studies on the central nervous system.     

Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides.

To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.     

Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization

We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehyde-fixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase antigenicity was better preserved in cryostat sections of PLP-fixed materials than in formaldehyde-fixed, paraffin-embedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.