a multiplex immunostaining method using primary antibodies from the same host species

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

Before staining, dewax and rehydrate formalin-fixed, paraffin-embedded slides with five-minute immersions in each solution as indicated. After the second distilled water immersion, place the slides in 275 milliliters of boiling sodium-citrate solution on the bottom of a pipette tip box with a lid, and place the box into a 700-watt microwave at 70% power for eight minutes.
At the end of the treatment, open the lid and allow the solution to cool to room temperature before washing the slides with three five-minute washes of fresh PBST in a Coplin jar. To block any nonspecific binding sites, after the last wash, shake the excess PBST from each slide and cover the slides with a sufficient volume of an appropriate blocking solution.
After 30 minutes at room temperature in a humidified chamber, shake the excess blocking solution from each slide, and add 250 microliters of the first primary antibody of interest to each sample. To prevent samples from drying out, the slides may be covered by a piece of paraffin film.
After an overnight incubation at four degrees Celsius in the humidified chamber, wash the slides three times with fresh PBST for five minutes per wash. Shake off the excess PBST, then, quickly cover each slide with 250 microliters of an appropriate secondary antibody solution for a one-hour incubation in the humidified chamber at room temperature.
At the end of the incubation, wash the slides three times in fresh PBST as demonstrated, and add 250 microliters of freshly prepared streptavidin horseradish peroxidase to each slide. After 30 minutes at room temperature, wash the slides three times in fresh PBST, then, shake off the excess PBST, and cover each slide with 250 microliters of freshly prepared fluorophore tyramide solution.
One minute later, submerge the slides in fresh PBST, followed by three two-minute washes in fresh PBST. After the last wash, apply a few drops of a 1-to-1 glycerol to PBS solution to the sections, and check for the presence of a fluorescent signal by fluorescence microscopy.
To strip the first antibody complex, place the antibody-labeled slides in 275 milliliters of boiling sodium-citrate solution for at least eight minutes as demonstrated. At the end of the treatment, open the lid and let the solution cool to room temperature, before washing the slides three times with PBST for five minutes per wash.
To stain for the second target protein of interest, after blocking for nonspecific binding is demonstrated, label each sample with 250 microliters of the second primary antibody of interest at four degrees Celsius overnight. The next morning, wash the slides three times for five minutes in fresh PBST per wash before covering each slide with 250 microliters of an appropriate secondary antibody solution for a one-hour incubation at room temperature.
At the end of the incubation, wash the slides three times in fresh PBST as demonstrated, and add 250 microliters of freshly prepared streptavidin horseradish peroxidase to each slide. To develop the signal for the second target protein of interest after three PBST washes, treat the slides with a fluorophore-conjugated tyramide in a different fluorescent spectrum.
To stop the tyramide signal development, submerge the slides into PBST then stain the samples with an appropriate nuclear dye.

a-multiplex-immunostaining-method-using-primary-antibodies-from-same

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Staining with the First Antibody
<ol>
	<li>Dewax and rehydrate formalin-fixed paraffin-embedded (FFPE) slides with 5 min allotted to each of the following steps: xylene or equivalent reagents 3x, 100% ethanol 2x, 95% ethanol 1x, 70% ethanol 1x, 50% ethanol 1x, and distilled water 2x. 
	NOTE: Slides should remain moist starting from this rehydration step until mounting in the final step.</li>
	<li>For optional antigen retrieval, place the slides in 275 mL of boiling sodium citrate solution (10 mM, pH = 6.0) for 8 min. To keep the solution boiling, place the slides flat on the bottom of a 14 x 9.5 x 9 cm3 (W x L x H) pipette tip box with a lid and microwave the solution on 70% power in a 700 W microwave oven for 8 min. Then remove the pipette tip box from the microwave oven, open the lid, and let the solution cool down at room temperature (RT) for at least 20 min.</li>
	<li>Transfer the slides into a Coplin jar containing PBST (phosphate-buffered saline with 0.1% polysorbate 20/80). Wash with PBST for 5 min 3x. If not immediately moving to the next step, store the slides at this step with PBST in a Coplin jar at RT for a few hours or at 4 &#176;C for 1-2 days if not immediately moving to the next step.</li>
	<li>To prepare the blocking solution use the normal serum of the secondary antibody&#39;s host species as the blocking reagent. Other commercial blocking reagents may also be used. If using normal serum employed for the study presented in this study, add 100 &#181;L of normal donkey serum to 4.9 mL of PBST. The blocking solution can be stored at 4 &#176;C for up to 3 days.</li>
	<li>For blocking, shake off the PBST from the slides and quickly cover them with sufficient blocking solution. Incubate the slides at RT in a humidified chamber for 30 min.</li>
	<li>Prepare the primary antibody solution (500 &#181;L for two slides) using the blocking solution to dilute the primary antibody to the desired concentration. The solution should be stored on ice until use.
	<ol>
		<li>For 3&#946;-hydroxysteroid dehydrogenase (3&#946;HSD), add 2 &#181;L of 3&#946;HSD antibody into 498 &#181;L of blocking solution.</li>
		<li>For tyrosine hydroxylase (TH), add 0.5 &#181;L of TH antibody into 499 &#181;L of blocking solution.</li>
		<li>For &#946;-catenin, add 1 &#181;L of &#946;-catenin antibody into 499 &#181;L of blocking solution.</li>
		<li>For 20&#945;-hydroxysteroid dehydrogenase (20&#945;HSD), add 1 &#181;L of 20&#945;HSD antibody into 499 &#181;L of blocking solution.</li>
		<li>For cytochrome P450 2F2 (CYP2F2), add 2 &#181;L of CYP2F2 antibody into 498 &#181;L of blocking solution.</li>
	</ol>
	</li>
	<li>Incubate with the primary antibody by shaking off the blocking solution from the slides, and quickly covering them with sufficient primary antibody solution. Incubate the slides in a humidified chamber overnight at 4 &#176;C. During incubation the slides may be covered by a small piece of paraffin film to prevent drying out.</li>
	<li>The next morning wash the slides with PBST for 5 min 3x.</li>
	<li>Prepare the secondary antibody solution (500 &#181;L for two slides) using the blocking solution to dilute the biotinylated secondary antibody to the desired concentration (e.g., 1 &#181;L of donkey anti-mouse antibody or donkey anti-rabbit antibody into 499 &#181;L of blocking solution). The solution should be stored on ice until use.</li>
	<li>Incubate with the second antibody by shaking off PBST from the slides and quickly covering them with sufficient secondary antibody solution. Incubate the slides in a humidified chamber for 1 h at RT. 
	NOTE: If endogenous biotin is a concern, use a peroxidase-conjugated secondary antibody instead of the biotinylated secondary antibody. Jump to step 1.14 if using a peroxidase-conjugated secondary antibody in step 1.10.</li>
	<li>Wash the slides with PBST for 5 min 3x.</li>
	<li>Prepare the horseradish peroxidase-conjugated streptavidin (SA-HRP) solution (500 &#181;L for two slides) by adding 0.5 &#181;L of SA-HRP into 499 &#181;L of PBS. The final concentration is 1 &#181;g/mL.</li>
	<li>Incubate with the SA-HRP solution. Shake off PBST from the slides and quickly cover the slides with sufficient SA-HRP solution. Incubate the slides in a humidified chamber for 0.5 h at RT.</li>
	<li>Wash the slides with PBST for 5 min 3x.</li>
	<li>Prepare the fluorophore-tyramide solution by diluting fluorophore-tyramide with its dilution buffer according to the manufacturer&#39;s instructions (e.g., 5 &#181;L of fluorophore-tyramide into 496 &#181;L of dilution buffer).</li>
	<li>Perform the signal development with fluorophore-tyramide by shaking off PBST from the slides and quickly covering the slides with sufficient fluorophore-tyramide solution. Incubate in a humidified chamber for 1 min at RT. The incubation time may be adjusted to obtain the desired fluorescence intensity. Stop the reaction by transferring the slides to a Coplin jar containing PBST. Wash slides with PBST for 3 min 2x.</li>
	<li>Signals may be quickly checked under a fluorescence microscope to confirm the result at this stage. If coverslips are not being used, apply a drop of glycerol:PBS (1:1) onto each section to keep the samples moist. Wash slides with PBST for 3 min 2x. Slides may be stored in PBST at 4 &#176;C for a few days before moving to the next step.</li>
</ol>
2. Strip the First Antibody
<ol>
	<li>Place the slides in 275 mL of boiling sodium citrate solution (10 mM, pH = 6.0) for at least 8 min. Keep the solution boiling by placing the slides flat on the bottom of a 14 x 9.5 x 9 cm3 (W x L x H) pipette tip box with a lid and microwaving the solution on 70% power in a 700 W microwave oven for 8 min. If a longer stripping time is preferred, it may be required to top off the buffer using a boiling sodium citrate solution to keep the slides immersed in buffer solution at all times. Remove the pipette tip box from the microwave oven, open the lid, and let the solution cool down at RT for at least 20 min.</li>
	<li>Transfer the slides into a Coplin jar containing PBST. Wash with PBST for 5 min 3x. If the next step if not performed immediately, store the slides in a Coplin jar with PBST at RT for a few hours or at 4 &#176;C for 1-2 days.</li>
</ol>
3. Stain with the Second Antibody
<ol>
	<li>Start from the blocking step and follow same procedures in step 1.5 through step 1.14. At the signal development steps (step 1.15 and step 1.16), use the fluorophore-tyramide in a different fluorescence spectrum. 
	NOTE: Slides can be stripped using this method several times to allow multiplex staining. The last antibody does not require fluorophore-tyramide as the reporter. A fluorophore-conjugated secondary antibody might be used to show signals from the last primary antibody.</li>
	<li>Incubate slides with 2 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) or Hoechst solution for 1 min at RT if nuclear counter staining is needed. Then wash the slides with PBST for 3 min 2x.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated donkey anti-mouse</td>
			<td>JacksonImmuno</td>
			<td>715-066-151</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Biotinylated donkey anti-rabbit</td>
			<td>JacksonImmuno</td>
			<td>711-066-152</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>BioLegend</td>
			<td>422801</td>
			<td>2 &#956;g/mL in distilled water</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>ECHO</td>
			<td>Revolve 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated streptavidin</td>
			<td>JacksonImmuno</td>
			<td>016-030-084</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Microwave oven, 700W</td>
			<td>General Electric</td>
			<td>JEM3072DH1BB</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-CYP2F2</td>
			<td>Santa Cruz,</td>
			<td>SC-374540</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-TH</td>
			<td>Santa Cruz,</td>
			<td>SC-25269</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>JacksonImmuno</td>
			<td>017-000-121</td>
			<td>2% serum in PBST</td>
		</tr>
		<tr>
			<td>Rabbit anti-20&#945;HSD</td>
			<td>Kerafast,</td>
			<td>EB4002</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-3&#946;HSD</td>
			<td>TransGenic,</td>
			<td>KO607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-TH</td>
			<td>NOVUS,</td>
			<td>NB300-109</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-&#946;-catenin</td>
			<td>Abcam,</td>
			<td>ab32572</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Streptavidin Horseradish Peroxidase (SA-HRP)</td>
			<td>JacksonImmuno</td>
			<td>016-303-084</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>TSA Cy3 Tyramide</td>
			<td>PerkinElmer</td>
			<td>SAT704B001EA</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>TSA Fluorescein Tyramide</td>
			<td>PerkinElmer</td>
			<td>SAT701001EA</td>
			<td>1:100 dilution</td>
		</tr>
	</tbody>
</table>

Source: Lyu, Q., et al. <a href="https://app.jove.com/v/60868">Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to perform multiplex immunostaining using primary antibodies from the same host species. In this method, an adrenal tissue section undergoes a two-step immunostaining process. Initially, the section is stained with antibodies specific for markers in the cortex, followed by stripping the bound antibody and restaining with antibodies specific for markers in the medulla.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Staining with the First Antibody

...

Take a fixed and permeabilized adrenal section comprising a cortex and a medulla. Treat it with a boiling acidic buffer, breaking the fixation-induced crosslinks for antigen unmasking.
Block the non-specific binding sites and introduce primary antibodies binding to cortex-specific markers.
Add biotinylated secondary antibodies targeting the primary antibodies and streptavidin-conjugated peroxidases binding to biotin.
Introduce a fluorophore-tagged tyramide and hydrogen peroxide.
The immobilized peroxidases utilize hydrogen peroxide to activate tyramide, which binds to nearby proteins, labeling the cortex.
Next, treat the section with the boiling buffer to strip the bound antibodies.
Introduce the same host species-generated primary antibodies targeting medulla-specific markers and biotinylated secondary antibodies.
Introduce streptavidin-conjugated peroxidases and a different fluorophore-conjugated tyramide to label the medulla.
Stripping the cortex-specific primary antibodies inhibits the reintroduced secondary antibody binding them, preventing cross-reactivity.
Apply a nuclear stain and perform imaging to visualize the distinctly stained cortex and medulla, confirming no antibody cross-reactivity.

21 visualizações. Um método de imunocoloração multiplex usando anticorpos primários da mesma espécie hospedeira

Vídeo: Um método de imunocoloração multiplex usando anticorpos primários da mesma espécie hospedeira - Experimento

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Dissecção assistida por transilluminação de etapas específicas do ciclo epitelial seminifero do rato para análises de imunostaining a jusante

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

JoVE Journal

Biologia do Desenvolvimento

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Snap o Chip para imunoensaios livre de Cross-reactivity e Spotter sanduíche multiplexado

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengenharia

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

Transcrição

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species