Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.     

Quantitative aspects of immunogold labeling in embedded and in nonembedded sections

We tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in the protein's structural conformation occur that would affect its immunoreactivity. In spite of a higher sensitivity observed for Ig-gold, we preferred to use protein A-gold in our system because of the low nonspecific labeling and the more precise antigen detection by the latter immunomarker.     

A method for using low-temperature embedding media for electron microscopy of cells grown on microporous supports.

Attempts were made to embed Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose microporous supports in Lowicryl, a medium designed for the retention of antigenicity for electron microscopic immunocytochemical studies. It was found that the membrane fragmented during the prescribed embedding procedure leading to loss of the cell sample. This necessitated the formulation of a new combination of fixative and dehydration agents which would allow: (i) preservation of membrane and cellular integrity; (ii) infiltration and embedding in Lowicryl; and (iii) detection of a specific antigen in thin sections. The cellular monolayer and organelle profiles were best preserved with glutaraldehyde fixation at room temperature followed by ethanol dehydration. Since the latter was carried out at temperatures attainable with an ice-salt bath, there was no need for a special ultralow-temperature apparatus. This procedure was applied to a MDCK cell clone that consisted of stable secretors of human growth hormone (hGH), as a result of being transfected with a plasmid containing the hGH gene. Thus, it was demonstrated that hGH could be detected by immunogold labeling of thin sections and localized to specific cellular structures. The procedure developed in this report is applicable to cells grown on two other supports and may be extended further.     

Optimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen.

We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.     

Use of anti-fluorophore antibody to achieve high-sensitivity immunolocalizations of transporters and ion channels.

We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.     

PC10 monoclonal antibody to proliferating cell nuclear antigen as probe for cycling cell detection in developing tissues

Proliferating cell nuclear antigen (PCNA), also referred to as cyclin, is an auxiliary protein to DNA-polymerase delta and a proposed marker of replicating cells. We have investigated the applicability and limitations of PC10 monoclonal antibody to PCNA in a cell kinetics study of developing human and rat tissues by immunocytochemical and flow cytometric techniques. Our data demonstrate that the epitope recognized by PC10 antibody is resistant to wax embedding, but sensitive to aldehyde fixation; conversely, alcoholic fixative solutions preserve the immunoreactivity to PC10. Tissue distribution, DNA content and bromodeoxyuridine uptake confirm that PC10-immunoreactive cells in alcoholfixed tissues are cycling (G1-, S- and G2-phases traversing) cells. It is concluded that the PC10 antibody can be regarded as a powerful tool to study cell kinetics and differentiation in developing tissues, provided that the tissue processing is adeguate.     

Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections

Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.     

An Improved Immunolabeling Method for Microtubular Cytoskeleton in Poplar (Populus nigra L) Free Nuclear Endosperm

We investigated immunocytochemical staining of microtubular cytoskeleton of free nuclear endosperm, a tissue which is particularly difficult to fix. This tissue requires fixation for 45 hr to preserve the integrity of the microtubular network after paraformaldehyde based fixation. Low glutaraldehyde concentration in the fixative and the ethanol dehydration retains β-tubulin antigenicity and the former improves preservation of tissue structure. An ethanol-free embedding method is recommended for immunocytochemical studies of ethanol sensitive target proteins.     

Use of dextran and post-primary antibody fixation in immunoperoxidase staining of fresh frozen tissue. Detection of immunoglobulin associated with squamous carcinomas of the head and neck

Use of unfixed fresh frozen tissue sections for immunocytochemical studies reduces the possibility of denaturation of antigenic determinants compared to formalin fixation and paraffin embedding procedures. However, tissue and cellular morphology can be extensively altered in the numerous application and washing steps with frozen tissue sections. We tested a number of buffer solutions and showed that the use of dextran-containing buffers and fixation by glutaraldehyde after primary antibody application preserves tissue morphology. The procedures described here are also applicable to ascertaining the presence of Fc receptors of leukocytes in sections of carcinoma tissues. The buffered dextran washes and post-primary antibody fixation method was used to demonstrate the presence of immunoglobulin associated with squamous carcinoma cells. The immunoglobulin was not removed by washing of tissue sections at 37 degrees C but could be removed by low or high pH buffer washes, suggesting that the immunoglobulin is bound in a specific manner.     

Freeze-drying technique in electron microscopic immunohistochemistry

Postembedding immunocytochemical labeling was performed on sections of rat neurohypophysis prepared by either freeze-drying, vapor fixation and Spurr resin embedding, or conventional aqueous fixation and Spurr resin embedding. Arginine vasopressin (AVP) and oxytocin (OXT) were immunolabeled with protein A-gold-anti-AVP and protein A-gold-anti-OXT complexes, respectively. The freeze-drying procedure (FD) resulted in excellent preservation of ultrastructure and greater antigenicity than the conventional procedure (Con). More gold particles were seen over secretory granules in FD sections than in Con sections. In addition, in FD sections, the gold label was restricted to secretory granules while in Con sections, both the granules and the extragranular axoplasm exhibited label. The two antigens in FD sections could be labeled simultaneously with protein A-small gold particle-anti-OXT complex and protein A-large gold particles-anti-AVP complex. In this way the two antigens were seen to be present in secretory granules within different axon terminals. Thus FD preparations should be useful for demonstrating the presence of multiple antigens in the same granules of nerve terminals.     

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.     

Polyester wax embedding and sectioning technique for immunohistochemistry

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.     

Different rhodopsin monoclonal antibodies reveal different binding patterns on developing and adult rat retina

We used a battery of 10 monoclonal antibodies directed against different identified peptide sequences within the carboxyl, transmembrane loop, and amino terminal regions of rhodopsin to label retinas from early postnatal and adult rats. Intensity of label, age of initial appearance of staining, and distribution of label varied depending on the antibody. Most antibodies showed detectable labeling at postnatal day 1, and were eventually observed binding to the cell bodies and the inner and outer segments of the photoreceptors. One amino terminal and two carboxyl terminal antibodies, however, showed no detectable labeling until postnatal day 5 and were only transiently detectable in the cell body region. These patterns cannot be explained by accessibility of binding site, binding affinity, fixation artifact, or crossreactivity. The results indicate that physiological and experimental parameters can alter the apparent immunocytochemical localization of conformationally active molecules such as rhodopsin. The results also suggest that rhodopsin can undergo light-dependent conformational changes in several different compartments within rat retinal photoreceptors before the time of eye opening.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling

We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.     

Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.     

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.     

An Improved Immunolabeling Method for Microtubular Cytoskeleton in Poplar (Populus nigra L) Free Nuclear Endosperm

We investigated immunocytochemical staining of microtubular cytoskeleton of free nuclear endosperm, a tissue which is particularly difficult to fix. This tissue requires fixation for 45 hr to preserve the integrity of the microtubular network after paraformaldehyde based fixation. Low glutaraldehyde concentration in the fixative and the ethanol dehydration retains β-tubulin antigenicity and the former improves preservation of tissue structure. An ethanol-free embedding method is recommended for immunocytochemical studies of ethanol sensitive target proteins.     

LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.