Below is a list of commonly used stains, often for different types of cells. All those listed can be used on fixed non-living cells and any that can be used on living cells are noted at the end of the description with the word "LIVE".
The image above shows how to draw a stain into a prepared slide. With the cover slip in place on top of the specimen, place a drop of stain on the edge of the cover slip. On the opposide side of the cover slip place a paper towel or cloth to draw the liquid out from the cover slip.
As the liquid is drawn out, the stain will be pulled in under the cover slip. Microscope Slide Staining Information Microscope cell staining is a technique used to enable better visualization of cells and cell parts under the microscope. By using different stains, a nucleus or a cell wall are easier to view. Most stains can be used on non-living fixed cells, while only some types of stain can be used on living cells.
Stem segments of 1 cm were collected from a region 5 cm distal of the rosette from inflorescence stems with an average height of 10 cm.
The same sections were observed with UV excitation [excitation filter: band pass — nm , emission filter: long pass nm ] after 1, 2, 7, and 21 days of incubation in paraformaldehyde and photographed with the exact same parameters Figures 2A—D. Day 1 corresponds to an incubation time of about 16 h see Material and Methods. A subtle increase of autofluorescence in the interfascicular fibers was already recorded by pixel measurement after 2 days of incubation in the fixative solution Figure 2E.
After 7 days of incubation in paraformaldehyde, the increase of primary fluorescence in the interfascicular fibers was clearly visible. The intensity of autofluorescence keeps increasing up to 21 days of incubation.
To ensure that the effect was due to paraformaldehyde and not its buffer solution, we have tested two solvents, a PEM buffer pH 6. We obtained equivalent result. In conclusion, to limit the intensity of primary fluorescence, it is recommended to store plant material in the fixative solution for the shortest amount of time. Figure 2. Primary fluorescence increases over time when transverse stem sections are stored in paraformaldehyde.
Transverse sections of A. The same sections cut with a vibratome were observed with UV excitation after 1 A , 2 B , 7 C , and 21 D days of incubation in paraformaldehyde. Using a long pass emission filter allows the visualization of primary fluorescence produced by the interfascicular fibers blue color and by the plastids located in the cortical parenchyma red color.
Comparison between micrographs A—D shows the increase of autofluorescence that occurs over time when the stem sections are stored in paraformaldehyde. The pixel measurement was carried out using ImageJ following the procedure detailed in Supplemental data S2. The micrographs underneath the graph represent the region selected for pixel measurement. For this example, the measurement was done in triplicate. Error bars: SD. Due to the high diversity of fluorescent compounds present in plants, a relatively strong autofluorescence is usually visible throughout the entire spectrum of wavelengths used in fluorescent microscopy.
This is a major concern when analyzing samples by immunomicroscopy since primary fluorescence overlaps with the emission of fluorescence produced by the fluorochromes used to detect cell wall epitopes see Figure 3. In some cases, it can be extremely difficult to visualize the presence of cell wall polymers because of primary fluorescence. As demonstrated in Figure 3 , the intensity of fluorescence depends of how the samples are prepared Figures 3A—C. Under the same conditions—which include the same emission and excitation wavelength [Excitation filter: Band pass — nm , Emission filter: Band pass — nm ] and the same time of exposure for capturing the image - the intensity of autofluorescence increases from the thinnest section Figure 3A to the thickest section Figure 3C.
Resin-embedded samples usually exhibit a low level of autofluorescence when observing the sample under the microscope through the eye-pieces. Consequently, no fluorescence is detected when photographing the sample with a short time of exposure Figure 3A.
By contrast, when applying the same parameters to the thicker wax-embedded samples, primary fluorescence is visible in the ring of interfascicular fibers and the xylem cells of the wax-embedded stems Figure 3B. The thickness of the samples and the embedding procedures are responsible for these differences of fluorescence intensity.
For the same surface, the thicker a plant sample is, the more it contains fluorescent compounds and thus the higher the intensity of fluorescence is. Moreover, the dehydration and infiltration steps performed during the resin and wax embedding lead to some loss of naturally fluorescent compounds. Figure 3. The detection of primary fluorescence and indirect immunolabeling relies on the preparation of plant samples.
Micrographs A—D show the primary fluorescence of equivalent transverse stem sections of A. This fluorescence is naturally emitted, for example, by phenolic compounds such as the lignin present in the interfascicular fibers if.
Micrographs E—H show the indirect immunolabeling of xylan, a cell wall polysaccharide recognized by the LM10 monoclonal antibody. The primary antibody binding is revealed with a secondary anti-rat monoclonal antibody coupled with a FITC fluorochrome. The intensity of both primary fluorescence and the artificial fluorescence resulting from the immunolabeling are proportional to the thickness of the sections.
As shown here, both type of fluorescence increase with section thickness. The sections obtained with a vibratome were used to demonstrate the effect of toluidine blue when the sections are excited under blue light.
D with, in this example, a minimal loss of epitope detection G vs. H , insets. The arrows in micrographs G,H highlight regions where the LM10 antibody bound weakly to cell walls when the sections were stained after immunolabeling. Changes in primary fluorescence will be more easily perceived in thick samples than in relatively or semi-thin sections where the autofluorescence can sometimes barely be visible.
Therefore, the use of vibratome is adequate for studying phenolic compounds such as lignin by fluorescence microscopy. By contrast, resin-embedded sections are preferable when the presence of primary fluorescence is not desired see Supplemental data S3.
There are several approaches to reduce the primary fluorescence when its presence is an issue. The primary fluorescence can be quenched by exposing the samples to intense light prior to labeling. However, photobleaching damages the samples and the technique is not convenient for immunolabeling of plant cell walls. It is possible to bypass the autofluorescence by carrying out immunogold labeling followed by silver enhancement and fuchsin couterstaining with observation of the samples by epi-polarized light microscope Bush and McCann, Yet, as it is easier, cheaper and faster, we stain the sections with toluidine blue.
This dye considerably reduces the detection of primary fluorescence under blue excitation Excitation: — nm Biggs, ; Leroux et al. As toluidine blue binds to lignin and feruloylated polysaccharides Smith and McCully, , the quenching of autofluorescence occurs regardless of how the plant material is prepared.
However, it is recommended to analyze the toluidine blue-stained sections under blue excitation using a band pass filter as emission filter. Toluidine blue possesses a phenolic ring that fluoresces when excited from ca. As illustrated in Figure 3 with LM10, an antibody directed to hemicellulosic xylan, the intensity of fluorescence resulting from immunolabeling depends on the method of sample preparation Figures 3E—G.
Under the same conditions, the occurrence of the LM10 epitope in the interfascicular fibers and xylem cells is weakly detected in the transverse stem sectioned with an ultramicrotome Figure 3E , the intensity of fluorescence is higher in the section prepared with a microtome Figure 3G and the fluorescent signal is saturated in the sections cut with a vibratome Figure 3F.
In our assay, the procedures of embedding and the sample thickness are the only two parameters that differ between the three types of sections. The difference of fluorescence detection likely relies on both parameters. In resin-embedded sections, the presence of the LR White resin prevents antibodies from penetrating into the sections so only the epitopes that are accessible at the surface can be detected. Sections embedded in wax or coated in agarose are not impacted by the presence of the embedding agent.
Consequently, in addition to the section surface X-Y-axis , the antibodies may also access the cell walls present in the inner surface Z-axis of these sections. It is reasonable to think that, in sections embedded in wax or coated in agarose, the antibody binding intensity increases with the thickness of the sections since the surface area, and thus the amount of epitopes accessible to the antibodies, are directly proportional.
Figure 4. The figure shows representative indirect immunolabeling of the LM16 pectic arabinan epitope in pith parenchyma cells from transverse stem sections of A. A Section embedded in LR White resin and cut with an ultramicrotome. B Section embedded in Steedman's wax and cut with a microtome. C Section coated in agarose and cut with a vibratome. The binding of the LM16 antibody varies depending on the sample preparation.
The arrows indicate the region of adhered cell walls where the LM16 epitope is abundantly detected. The insets at 2x scale highlight that a reduced antigenicity of the sample can provide better details on the fine localization of the epitopes.
In the insets, the epitope is seen to be present in the primary cell walls and absent from the middle lamella. The micrographs were taken with different times of exposure that were, for each method of sample preparation, determined based on a signal to noise ratio negative controls are shown in Supplemental data S4.
The shortest and longest times of exposure were taken for the sections cut with a vibratome and an ultramicrotome, respectively. Figure 5. The efficiency of enzymatic pre-treatment depends of the sample preparation. The figure shows the impact of pectate lyase pretreatment on indirect immunolabeling of the LM19 homogalacturonan and the LM21 mannan epitopes in pith parenchyma cells of transverse A.
The presence of the LM19 homogalacturonan epitope is ubiquitous in the walls of pith parenchyma cells and the degradation of homogalacturonan with pectate lyase results in a severe reduction of LM19 detection and an increased detection of the LM21 mannan epitope.
The immunolabeling obtained after enzymatic pretreatment showed nuances of enzymatic efficiency between the different methods of sample preparation. A,B,G,H Section embedded in resin and cut with an ultramicrotome. C,D,I,J : Sections embedded in wax and cut with a microtome. E,F,K,L : Sections coated in agarose and cut with a vibratome. A—F Representative sections immunolabeled with the LM19 homogalacturonan antibody.
G—L : Representative sections immunolabeled with the LM21 mannan antibody. The parameters used to capture these micrographs were identical between the pre-treatments. However, the time of exposure differed for both antibodies and for each method of sample preparation. The arrows in B highlight regions where residual traces of the LM19 epitopes are detected after pectate lyase pre-treatment. To avoid misleading the readers with this comparative assay, we must emphasize that what is important when performing immunolabeling is to ensure that the same times of exposure are applied to both immune- and control sections, regardless of which method is used see, for example, Figure 6.
In Figure 3 , the same time of exposure was used for each micrograph to illustrate the difference of fluorescence intensity that occurs in between the three methods of sample preparation.
However, since the resin-embedded sections show little if any primary fluorescence Figure 3A , it is not problematic to apply longer exposure times see Supplemental data S3 to achieve images that are comparable in brightness to the microtome or the vibratome sections shown here in Figures 3F,G , respectively. Likewise, for the sections cut with a vibratome, shorter time of exposure can be used to reduce the primary fluorescence and avoid a saturation of the fluorescent signal Supplemental data S3.
Figure 6. Impact of the sample preparation on indirect immunolabeling after alkaline pre-treatment. Transverse stem sections of A. As represented in this figure, the alkaline pre-treatment results in an increase of LM18 detection regardless of the how the samples were prepared. A,D Section embedded in resin and cut with an ultramicrotome. B,E Sections embedded in wax and cut with a microtome. C,F Sections coated in agarose and cut with a vibratome. A—C were incubated with phosphate buffer pH 7.
However, the time of exposure differed for each method of sample preparation. When the sections are stained with toluidine blue, the accumulation of green fluorescence emitted from both the plant material and the fluorescent probe is diminished.
The post-immunolabeling stain with toluidine blue greatly facilitates the distinction between the fluorescence produced by the labeling from the primary fluorescence Figures 3C,F vs. Figures 3D,H. However, this easy method of autofluorescence quenching may also lead to a loss of information. For instance, the occurrence of cell types weakly detected in unstained sections may sometime be barely or no longer visible in sections stained with the dye.
Such loss of detection can be observed when comparing micrograph Figures 3G,H in the regions highlighted by arrows and magnified in the insets. The reduction of detection is associated with the binding of toluidine blue to lignin and feruloylated polysaccharides and varies with the targeted epitopes as well as the source of plant material.
Prior to using it routinely, it is thus important to evaluate the effect of a post-immunolabeling stain with toluidine blue and to determine how it can impact the results.
Most fluorochromes attached to secondary and tertiary antibodies are prone to photobleaching. We have particularly noticed this when using fluorescein isothiocyanate FITC. To bypass the sensitivity of the fluorophore, immunolabeled samples can be soaked in protective solutions that prevent a rapid photobleaching. Alternatively, stable and robust fluorophores such as Alexa Fluors can be used in absence of antifadent solution e.
The technique of embedding can influence how epitopes are detected in plant material, not only in term of fluorescence intensity but also in term of localization.
How the methods of sample preparation impact on the immunolocalization varies with the epitope. Usually, immunolabelings show none or subtle differences of epitope occurrence between sections embedded in acrylic resin or wax or coated in agarose. For instance, in the transverse A. The LM19 homogalacturonan epitope detection in the parenchyma cells of A. Nevertheless, the loss of detection can sometimes be drastic for other epitopes.
This is exemplified in Figure 4 with the LM16 arabinan antibody. As previously reported Verhertbruggen et al. Embedding in wax Figure 4B results in a more abundant detection of the LM16 epitope than what is observed in the resin section.
Albeit weak, the epitope occurrence is visible all around most cell corners. The absence of LM16 epitope from the middle lamella can easily be observed in the wax-embedded section see inset in micrograph B. In the non-infiltrated sections, the arabinan epitope is consistently detected all around the cells and the overall intensity of fluorescence is high Figure 4C. Overall, the abundant detection of the LM16 arabinan epitope in the adhered walls of the cells and its absence from the middle lamella are visible regardless of the method of sample preparation used.
It is to note that, when looking through the eye-pieces, these observations are less easily noticed in sections cut with a vibratome than in sections cut with an ultramicrotome or a microtome. In this Figure, we have used different times of exposure to highlight in each type of sections where the LM16 epitope is immunolocalized. Severe loss of detection that is related to the method of sample preparation has already been reported for the LM7 epitope Willats et al.
In absence of pre-treatment, the LM21 epitope can only be detected in the parenchyma cells of A. If the thickness of the samples was the only parameter to affect its detection, the LM21 epitope would have been expected to still be visible in the microtome sections. This was neither observed here or by Marcus et al. Altogether, these results strongly suggest that the procedure of embedding can affect the preservation of certain epitopes.
We do not know what exactly causes the loss of carbohydrate epitopes but it is possible that they are extracted either by water or by alcohol and altered by the changes of pH that occurs during the steps of alcohol dehydration and infiltration.
Bypassing dehydration and infiltration preserves the antigenicity of the sections and, consequently, sectioning plant material with a vibratome allows obtaining the best vision of what the real epitope occurrence in muro is. However, vibratome-sectioning is not the most convenient technique when the aim is to inspect the in fine occurrence of epitope. Extra precautions must be taken to ensure that what is seen does not come from artifacts resulting from the thickness of the sections.
Resin-embedded sections present the advantages that they can be used both for light and electron microscopy. This is obviously of a great advantage when the aim is to gain knowledge on the ultrastructure of plant cell walls. However, for light microscopy, the loss of antigenicity and the cell wall alterations that can result from the embedding in resin or the presence of the resin itself are disadvantageous.
The wax-embedded sections also endure damages during the steps of dehydration and infiltration but, as the sections are dewaxed, the accessibility to the epitopes is not directly affected by the presence of the polyester.
For example, hematoxylin is a stain that turns cell nuclei blue. When used in conjunction with eosin, which turns the other parts of the cell red or pink, it provides a stronger contrast and makes the nuclei easier to differentiate.
PAP smears and blood marrow samples are easier to examine when these two stains are used together. Gram's Stain: Hospital workers use Gram's stain to identify harmful bacteria. This is actually a series of colorants that have different effects on different types of bacteria and give doctors an important diagnostic tool.
Gram's stain is a three-part process. In the first, Hucker's crystal violet is added, which stains all bacteria a uniform violet color. In the next stage, iodine stain is added, which causes the color to adhere to Gram-positive cells, which are primarily Staphylococcus and Streptococcus.
The stain is washed away, leaving the Gram-positive cells with a distinct violet color; then a third stain, Safranine O, is introduced to enhance the contrast between the Gram-negative bacteria and the rest of the material in the slide.
When preparing a specimen on a slide, you can dry-mount or wet-mount it, you can slice it into a thin section or you can smear it.
This is then followed by filtering papanicolaou staining system reagents before use. A 50 for two minutes,. Pusiol et al. CytoJournal 5 Acid fuchsin is a magenta red acid dye that is largely used for plasma staining whereas basic fuchsin is a magenta basic dye largely used to stain the nucleus. The technique is also referred to as acid fast staining. The acid fast bacteria have a waxy substance mycolic acid on their cell wall that makes them impermeable to staining procedures.
The term acid fast is used since they resist decolourization with acid alcohol. Carbol fuchsin , the primary stain contains phenol, which helps solubilize the cell wall whereas heat is used to increase the penetration of the stain. On using alcohol to decolorize, cells will be decolorized except for acid fast ones. Methylene blue is used as the counterstain to any cell that was decolorized.
Preparation of carbol fuchsin by mixing two solutions:. Solution 1- 0. Solution 2- 5 grams phenol and 90 ml of distilled water,. A photomicrograph of Mycobacterium smegmatis pink and Micrococcus luteus blue x magnification. Mycobacterium smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink.
Micrococcus luteus is not acid-fast, loses the carbol fuchsin during decolorization, and is counter-stained with methylene blue. This is a Romanowsky type of metachromatic stain that is prepared by mixing specially treated methylene blue dye with eosin. The acidic portion of the stain unites with the basic components of the cells such as hemoglobin, and thus they are referred to as eosinophilic and are stained pink or red.
The acidic components of the cell, such as the nucleic acids on the other hand take the basic dye and stain blue or purple. PH has to be controlled using a buffer of 6.
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