What Do We Do After the Second Incubation Why Do We Wash the Wells Again

Post-translational Modifications That Modulate Enzyme Activity

Monica Kruk , ... Laurie L. Parker , in Methods in Enzymology, 2019

3.3.4 Enzyme-linked immunosorbent assay

1.

Prepare primary and secondary antibody solutions. Dilute 4G10 antiphosphotyrosine primary antibody 1:5000 in blocking buffer (described in Section 3.3.1). Dilute anti-mouse HRP-conjugated secondary antibody 1:1000 in blocking buffer.

2.

In the streptavidin plate: incubate wells to be used for analysis with 100 μL of blocking buffer with gentle shaking at 500 RPM for 1 h.

3.

For each sample, add 100 μg of lysate directly to the 100 μL blocking buffer already in the wells, and bring all wells to an equal volume with a minimal amount of urea lysis buffer. Incubate at room temperature with gentle shaking at 500 RPM for 1 h.

Tip: Synthetically phosphorylated substrate can be used as a positive control for ELISA detection conditions by including triplicate incubation of synthetic phosphopeptide (~ 200 pmol/well) in blocking buffer in control wells alongside the lysate incubation. To provide controls for antibody specificity, also include triplicate incubation of synthetic substrate peptide (same as used as the substrate in the assay as described in step 2 of Section 3.3.3 ), which should not give any signal from the 4G10 antibody. The "no substrate" control from the assay in Section 3.3.3 will also serve as an antibody specificity control since it contains no phosphorylated Abl substrate peptide.

4.

Remove supernatant from wells (see tip below) and wash with 250 μL blocking buffer per well, pipetting up and down 15 times. Discard wash by aspiration or pouring into waste container, taking care not to cross-contaminate wells. Repeat for a total of three washes.

Tip: Supernatant containing 100 μg lysate can be collected and stored at 4 °C until analysis is complete, or discarded. After each step, ensure liquid is completely removed from wells as best as possible, but do not leave the wells dry for long periods of time.

5.

Add 100 μL primary 4G10 antibody to each well and incubate at room temperature with gentle shaking at 500 RPM for 1 h. Empty wells by pouring into waste and wash three times as described in step 4.

6.

Add 100 μL secondary HRP-conjugated antibody to each well and incubate at room temperature with gentle shaking at 500 RPM for 1 h. Empty wells by pouring into waste and wash three times as described in step 4.

7.

While secondary antibody is incubating with wells, prepare Amplex Red developing solution (described in Section 3.3.1). After washing away excess secondary antibody, add 100 μL developing solution to each well and incubate for 30 min in the dark.

Tip: Plates with developing solution can be protected from light by incubating in a drawer or wrapped in foil.

8.

Use a plate reader to measure Amplex Red fluorescence signal according to manufacturer's instructions (excitation between 530 and 560 nm, emission 590 nm).

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Early diagnosis with ultrasensitive ELISA

Etsuro Ito , ... Satoshi Watabe , in Advances in Clinical Chemistry, 2021

2 Protocol

Briefly, a sandwich ELISA using primary and secondary antibodies for the antigens, i.e., target proteins, was coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling [8,11]. In the forward reaction, an androsterone derivative, 3α-hydroxysteroid, is produced via hydrolysis of 3α-hydroxysteroid 3-phosphate by alkaline phosphatase (ALP, EC. 3.1.3.1) linked to the secondary antibody. The 3α-hydroxysteroid is subsequently oxidized to a 3-ketosteroid via 3α-hydroxysteroid dehydrogenase (3α-HSD, EC. 1.1.1.50) using thio-NAD as a cofactor. In the reverse reaction, the 3-ketosteroid is reduced to 3α-hydroxysteroid by 3α-HSD using NADH as the cofactor. During cycling (Fig. 1), thio-NADH can be optically measured at 405 nm without any interference from other cofactors.

Using a microtiter plate-based format, a solution containing the primary antibody was added to each well. Following incubation, the wells were washed to remove unbound antibody. The specimen and ALP-conjugated secondary antibody were then added to each well. Following incubation, the wells were washed and the cycling solution containing thio-NAD and NADH (cofactors), 17β-methoxy-5β-androstan-3α-ol 3-phosphate (primary substrate) and 3α-HSD (cycling enzyme) was added. The reaction was optically monitored for the generation of signal thio-NADH at 405 nm.

Thio-NADH signal intensity was expressed as:

$a \times b \times \sum_{k = 1}^{n} k = a \times b \times \frac{n (n + 1)}{2}$

wherein, a is the turnover ratio of ALP per min, b is the cycling ratio of 3α-HSD per min and n is measurement time in min. Signal generated at 405 nm was normalized to signal at 620 nm.

This ultrasensitive approach required minimal equipment other than a relatively inexpensive microplate reader thus making it feasible for most laboratories. Derivatives of androsterone were also assessed in this reaction format. Of these, 17β-methoxy-5β-androstan-3α-ol 3-phosphate demonstrated the most efficient hydrolysis rate via ALP and highest reaction efficiency, i.e., highest turnover number for 3α-HSD. This ultrasensitive ELISA was subsequently used in a number of clinical applications as described below.

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The Role of Macrophages in Antibody Responses in Vitro

Carl W. Pierce , Judith A. Kapp , in Immunobiology of the Macrophage, 1976

B Advantages and Disadvantages of Tissue Culture Systems

These two culture systems support the development of primary and secondary antibody responses with approximately equal efficiency. PFC responses that develop in culture are comparable in magnitude, kinetics, Ig class, and dependence on antigen dose to responses in intact animals during the first 7 days after immunization (Claman and Mosier, 1972; Pierce, 1974). This parallelism between responses in vivo and in vitro has been observed for most antigens that have been studied thoroughly. It also has been the general experience that those antigens that stimulate poor primary responses in vivo also stimulate poor primary responses in vitro. Since culture systems are closed systems and one has more precise control of the components of these systems than is possible in vivo, they are well suited for investigations of the critical factors in the induction of the antibody response. For example, the numbers, types, and immune reactivities of cells from the immune system that are added to the cultures and the interactions among these cells can be rather precisely controlled. The concentration and physical form of the antigen and the interaction of the various types of cells with antigen in the cultures can also be controlled. Furthermore, specifically reactive cells or reagents, such as antimetabolites, antibodies to antigen, or membrane molecules on the responding cells, can be added to the system at defined times in known numbers or concentrations (Pierce, 1974). Although antibody responses in vitro are comparable in many respects to in vivo responses, and although tissue culture systems offer many advantages in experimentation not possible in vivo, one must be extremely cautious when extrapolating from one system to the other, and one should avoid making dogmatic statements based on results from one system which have not been confirmed in the other.

Tissue culture systems are not without disadvantages. Spleen cells or partially purified T and B cells from mice are most commonly used in these culture systems; lymphoid cells from rabbit, rat, and chicken have been used successfully only by a few investigators. Most of the data we will discuss have been derived from cultures of mouse cells. However, even with mouse lymphoid cells, considerable strain to strain variation exists, and no amount of cajoling or uttering of incantations, friendly or hostile, can convince spleen cells from some strains of mice to respond faithfully on a regular basis. Cell survival in these cultures is not good; only approximately 25% of the initial cells survive for 5 days. Thus, we are dealing with a dying system, and the effects of products released from dead or dying cells on the surviving cells are still largely unknown. The tissue culture environment itself is artificial and may not provide optimal conditions in terms of nutrients. In addition, fetal bovine serum, not required for antibody responses in vivo, is a necessary ingredient of the culture medium. Fetal bovine sera vary in their capacity to support antibody responses in vitro; some are nonsupportive, and others are nonspecifically cytotoxic. This variability is an annoyance that could readily be done without. Also absent from tissue culture systems are the multitude of in vivo homeostatic mechanisms, such as the influences of circulating immunocompetent cells, the influx of virgin precursor cells from central lymphoid tissues, and the feedback regulation by other stimulated lymphocytes and previously synthesized antibodies, all of which obviously affect development and expression of antibody responses. Last, antibody responses in tissue culture systems are subject to a variety of maladies that we call, in all seriousness, gremlins (C. W. Pierce and J. A. Kapp, unpublished observations, 1975). These include the propensity of the investigator to put fingers in the cultures or to drop the cultures and various acts of God and man, such as the tides, phases of the moon, rocket launches, and the changes of the seasons. Nevertheless, tissue culture systems have been extremely useful for investigating many facets of the antibody response; the function of macrophages has been one of these facets.

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Immunofluorescence

Sonali Joshi , Dihua Yu , in Basic Science Methods for Clinical Researchers, 2017

Blocking to Limit Nonspecific Antibody Interactions

IF staining is enhanced by blocking nonspecific interaction of the primary and secondary antibodies with the biological sample. Nonspecific binding may result from inappropriate binding of the antibody to nonantigen molecules by excess unreacted aldehyde, trapping of the antibody in hydrophobic structures or by low-affinity polyclonal antibody binding to nonspecific molecules. Incubating the sample in a protein solution prior to incubation with the primary antibody prevents these nonspecific interactions. The sample is incubated with blocking agents such as bovine serum albumin, milk, and serum. Note: Serum is generally the reagent of choice for IF staining. It is important to note that the blocking serum should be obtained from a species distinct from the species in which the primary antibody was raised. If performing indirect IF, the blocking serum should belong to the species in which the secondary antibody was developed. As a lot of the widely used secondary antibodies are raised in goat, goat serum is a common choice for the blocking step. For blocking, incubate the sample in a 5% serum solution (in (1X) PBS with 0.05% Tween-20 or 0.05% Triton X-100) for 30 minutes to an hour. Commercially available blocking buffers containing highly purified single proteins or proprietary protein-free compounds may also be used for blocking.

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Immunocytochemical Analysis of Human Stem Cells

Jamison L. Nourse , ... Lisa A. Flanagan , in Human Stem Cell Manual (Second Edition), 2012

Fc Receptors in Sample

■: Problem: Fc receptors expressed by cells non-specifically bind primary and secondary antibodies. This is particularly problematic for tissues that have been damaged and contain activated immune cells.
■: Solution: Use Fab preparations for detection rather than whole antibodies or block using unconjugated Fc fractions that match both primary and secondary antibody preparations.

Note

When using Fab fragments for detection, the secondary antibody must be one that recognizes a Fab fragment. Typically, the secondary antibody used will recognize the light chain rather than heavy chain and one must take care to determine the class of light chain present in the Fab fragment (i.e. either kappa or lambda light chain).

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Expansion Microscopy for Cell Biology

Daphne Jurriens , ... Lukas C. Kapitein , in Methods in Cell Biology, 2021

3.1.2 Immunofluorescence

The expansion protocol is compatible with standard immunofluorescence protocols. Here we use primary and secondary antibodies to stain different types of microtubules. When imaging cytoskeleton proteins, the addition of an extraction step before cells fixation is recommended. The extraction step includes exposure to Triton X-100 and glutaraldehyde at low concentrations for pre-permeabilization of the cell membrane, removal of the soluble tubulin pool and stabilization of the polymerized cytoskeletal filaments ( Auinger & Small, 2008; Kapitein et al., 2010; Korobova & Svitkina, 2008).

1.: Pre-warm 500 μL of extraction and 500 μL of fixation buffer to 37 °C per coverslip. Remove the medium from the cells and gently add extraction buffer for 1 min. Remove extraction buffer and gently add fixation buffer for 10 min.
2.: Remove fixation buffer and wash the sample 3 times with 500 μL PBS. Start with a quick wash by adding the PBS followed by aspirating and immediately continuing to the second wash. For second and third wash steps wait 5 min.
3.: A permeabilization step is added to ensure all membranes are completely dissolved and the antibodies can completely access the structure of interest. Remove the PBS wash and add 500 μL permeabilization buffer for 10 min. After permeabilization, perform three wash steps with 500 μL PBS as described previously, starting with a quick wash followed by two washes of 5 min.
4.: Block the sample with 500 μL of blocking buffer for at least 45 min at room temperature to reduce unspecific binding of the antibodies.
5.: After blocking, incubate the samples with primary antibodies specifically targeting the structure of interest. As expansion dilutes the signal, we increased the concentration of the primary antibodies approximately 5× compared to standard staining procedure. For example, for Mouse monoclonal anti alpha tubulin we typically use 1/1000 dilution. We increase this to 1/200 dilution for expanded samples. Dilute the primary antibodies in blocking buffer and incubate the samples either 2 h at room temperature or overnight at 4 °C.
6.: After incubation with the primary antibodies, wash the cells 3 × with 500 μL PBS. The first wash is quick, followed by two 10 min washes. Subsequently, incubate the cells for 1 h at room temperature with secondary antibodies in blocking buffer. The concentration of the secondary antibodies is increased 2× compared with standard staining procedure. For example, Alexa Fluor 594 Goat Anti-Rat IgG we typically dilute 1/500, for expanded samples we dilute this 1/250. After incubation wash the cells again with PBS as described before, with one quick wash followed by two 10 min washes.
7.: To crosslink fluorescence labels to the gel, the cells are post-fixed with AcX or glutaraldehyde after staining (see these two options in Section 2). For AcX crosslinking, add 1000 μL of post-fixation buffer to cells and incubate overnight at room temperature. For glutaraldehyde crosslinking, incubate for 10 min. The last option requires shorter time, but we found that it provides less continuous microtubule staining preservation (Fig. 1F and G). While general intensity stays roughly the same, microtubules are labeled less continuously. This can be seen in the representative image (Fig. 1F) and the reduced standard deviation of the mean intensity (Fig. 1G).
8.: Following post-fixation, wash the cells 3× with 500 μL PBS for 5 min. Optionally fiducial markers can be added at this stage to make it easier to navigate and locate the sample during imaging. We found that plastic fluorescent beads (see Section 2) non-specifically stick to the surface of coverslips and cells and are retained in the expanded gel.

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The Unfolded Protein Response and Cellular Stress, Part A

Šárka Lhoták , ... Richard C. Austin , in Methods in Enzymology, 2011

5.3.3 General protocol

Particulars for each antibody, such as antigen retrieval, blocking serum, and primary and secondary antibody information are listed in Table 2.1.

1.: Sections are dried at room temperature overnight.
2.: Sections are deparaffinized in three changes of xylene, 10 min each.
3.: Three changes of 100% ethanol, 1 min each
4.: Endogenous peroxidase block: 60 ml methanol + 1 ml 30% H₂O₂ + 4 drops of concentrated HCl, 10 min
5.: Wash in 70% ethanol, then distilled water three times
6.: Perform an antigen retrieval if required (Table 2.1 and Section 5.3.3.1).
7.: Rinse with Tris buffer.
8.: Move slides to the humidity chamber. Slides are now in horizontal position; avoid drying.
9.: Block with 5% normal serum (see Table 2.1).
10.: Flick off excess blocking serum; wipe a rectangular area around the tissue with Kleenex. This will keep the primary antibody from spreading over the slide and drying.
11.: Incubate with primary antibody diluted in the 5% normal serum, for 1–2 h, or overnight at 4 °C.
12.: Wash with Tris buffer 2×.
13.: Incubate with biotinylated secondary antibody for 30 min. ¹
14.: Wash with Tris buffer 2×.
15.: Incubate with streptavidin-peroxidase, diluted 1 drop in 2 ml of Tris buffer, for 10 min.
16.: Wash with Tris buffer.
17.: Wash with distilled water.
18.: Prepare Nova Red solution according to manufacturer's directions.
19.: Incubate for 2–15 min, observing the reaction through microscope on the positive control or a section that is expected to be positive.
20.: Wash with distilled water.
21.: Counterstain with Gills hematoxylin No. 3, 30 s.
22.: Wash with tap water.
23.: Dehydrate in two changes of 100% ethanol and two changes of xylene. Use different xylenes and ethanols than for deparaffinizing. Avoid lower concentration of ethanol, go directly to 100% ethanol, and move quickly through ethanols and xylenes. Nova Red staining sometimes tends to disappear in ethanols.
24.: Mount with Permount.

5.3.3.1 Antigen retrieval techniques

Heat-induced epitope retrieval (HIER)

•: Preheat appropriate buffer in a plastic slide container in rice steamer for 25 min
•: Place slides in the preheated buffer, let steam for 30 min
•: Remove the container from rice steamer, let cool down in the same solution for additional 30 min

Citric buffer, pH 6.0

•: Dissolve 2.3 g of citric acid in 1000 ml of distilled water.
•: Adjust pH to 6.0 with 2 N NaOH (~ 13 ml)

Retrieve-all Antigen Unmasking System 2: Basic pH 10, 1× (SIGNET) #SIG-31922, Covance

Protease digestion

•: Dissolve 0.025 g of protease (Streptomyces griseus, #P6911, Sigma) in 50 ml of PBS
•: Treat slides for 5 min at room temperature
•: Wash with PBS, then Tris buffer

Triton X treatment

0.1% Triton X in PBS, incubate for 10 min

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Guide to Protein Purification, 2nd Edition

Alice Alegria-Schaffer , ... Krishna Vattem , in Methods in Enzymology, 2009

4.3.4 Antibodies

Not only is the affinity of the primary antibody for the antigen important, but primary and secondary antibody concentrations also have a profound effect on signal intensity. Too much HRP on the blot can be caused by either primary and secondary antibody concentrations or both. Minimal primary antibody is advantageous, as it promotes target-specific binding and low background.

If a blot failed to generate an adequate signal, removing all detection reagents (stripping) from the blot and reprobing with either a different primary antibody or different concentrations of antibodies often conserves valuable sample and time; however, insufficient stripping can leave active HRP on the blot that will produce a signal. Applying substrate on the stripped blot and subsequent detection will reveal if active HRP remains on the blot. Also, an abundance of inactive HRP molecules not removed by stripping can inhibit the primary antibody from binding to the target. Stripping and reprobing blots is an effective method to gain information about a specific system, but it is not a definitive way to determine the optimal system parameters.

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Methods in Extracellular Matrix Biology

Douglas R. Keene , Sara F. Tufa , in Methods in Cell Biology, 2018

2.5.1 "En bloc" Immunolabeling of Tissue

By far the most common immunocytochemical method used in our laboratory is described as the "en bloc" immunolabeling protocol. This method relies on diffusion to transport primary and secondary antibody into the bulk of fresh, unfixed tissue (Fig. 17A and C). At acquisition, storage, and handling, the tissue is kept moist with DMEM and trimmed with disposable double-edge razor blades so that one dimension of the tissue block is less than or equal to 1 mm. As much as possible, trim hair and fat from the tissue as it will cause it to float in subsequent solutions. The tissue is rinsed in borosilicate tubes filled with DMEM, then incubated overnight at 4°C in a 150 μL primary antibody diluted 1:5 or 1:10 in DMEM. Rarely will the protocol be successful if antibody is further diluted. Importantly, we use ~ 4-mL borosilicate tubes for antibody incubation. Be sure that the tissue sinks to the bottom of the tube during all incubations; it will likely be necessary to tap the bottom of the tube on a metal surface several times to dislodge air bubbles which will otherwise cause the tissue to float. During incubation in primary and secondary antibody, tissues are gently agitated on the platform of a small rotator (Sarstedt Sarmix GM1), which easily fits in a small refrigerator. Following incubation in primary antibody, the samples are rinsed three times over a minimum of 4 h in DMEM, then incubated in gold conjugated secondary antibody diluted 1:3 in DMEM, again on a rotator overnight at 4°C. Although we typically include a secondary antibody–gold conjugate, on occasion we use primary antibody directly conjugated to colloidal gold, resulting in more intense and specific localization (Keene, Oxford, & Morris, 1995). The size of the colloidal gold conjugate is critical. "Ultrasmall" (subnanometer) gold conjugates will diffuse into the tissue farthest and will also penetrate basement membranes (Fig. 17C); 5-nm will not penetrate basement membranes; 10-nm colloidal gold penetrates only a few microns into loose connective tissues, and 20-nm conjugates are not useful at all. Gold conjugates smaller than 2-nm need to be "enhanced," a protocol designed to precipitate metal onto the ultrasmall gold particulate, effectively making it visible in the TEM (Fig. 17C). We use a gold intensification kit (Nanoprobes, Inc.) as follows:

1.: Tissues are confined within 4-mL glass borosilicate tubes and rinsed in DMEM.
2.: Tissues and all gold enhancement components are cooled on ice.
3.: Equal drops of component A and B are combined and chilled on ice for 15 min, after which equal parts of C and D are added to solution AB.
4.: Tissues are incubated in the ABCD solution for 15 min on ice, after which the tubes are transferred to a 25°C water bath for exactly 5 min. The idea here is that the gold enhancement solutions are allowed to diffuse through the tissue at a low-enough temperature to inhibit any precipitation of gold. Warming the tissue quickly after low-temperature diffusion allows the enhancement to proceed uniformly within the tissue volume. This may also be accomplished in an EM-grade microwave oven, 1 min at 100 watts followed by another minute at 100 W.
5.: Samples are then rinsed in three quick changes of ice cold DMEM.
6.: Samples may then be immersed in primary fixative, rinsed, then immersed in secondary fixative including osmium.

After the tissue is embedded, trim the block so that your section includes blank epoxy bordering at least one edge of the tissue. It will only be at a free-cut edge of the tissue where the secondary antibody–gold conjugate can be imaged; gold will not penetrate throughout the bulk of the tissue.

With dense tissues, it may be necessary to solubilize some components of the matrix in order to penetrate the tissue with antibody. Cartilage and bone both have inherent high density, not only due to closely packed collagen fibrils and mineralized matrix (in bone) but also due to a high content of proteoglycan. Successful en bloc immunoelectron microscopy may therefore depend on selectively solubilizing the proteoglycan network, allowing diffusion of antibodies into the matrix. To allow penetration into intact, unfixed cartilage, the tissue may be incubated in chondroitinase ABC (diluted 0.25 units/mL) at 4°C overnight, and then rinsed in buffer prior to exposure to primary and secondary antibody.

A hybrid example of the en bloc method is to expose epitopes within cryostat sections cut from tissue frozen in Tissue-Tek OCT. OCT may be solubilized from the thawed tissue by rinsing in DMEM, and then the cryostat section may be exposed to antibody en bloc. During embedding the section may be flattened with the weight of an epoxy blank so that sections may be cut enface to the thicker cryostat section. The protocol will result in poor cellular ultrastructure (due to ice crystal damage) but by the same mechanism of ice crystal expansion the tissue may be "loosened" enough to allow diffusion of primary and secondary antibody. Poole and coworkers exposed proteoglycan, link protein, and collagens on 5-μm cryostat sections using an immunoperoxidase procedure, after which the tissue was fixed in glutaraldehyde and osmium tetroxide, and then embedded in epoxy. Indeed, ice damage resulted in poor cellular structure; however, antibody labeling of the proteoglycan aggregates was intensely successful (Poole, Pidoux, Reiner, & Rosenberg, 1982).

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Passive immunization

E. Richard Stiehm , Margaret A. Keller , in Vaccines (Sixth Edition), 2013

Bacterial respiratory infections

Respiratory tract infections secondary to group A Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitides, Mycoplasma, Chlamydia pneumoniae, and multiple viruses are more frequent and severe in patients with primary and secondary antibody immunodeficiencies. ^4, ^6, ^15–17 These infections can be markedly reduced by regular administration of immunoglobulin. Furthermore, specific antisera to some of these organisms were used in the early 1930s for the treatment of severe infections (e.g., meningitis, epiglottitis) even after the introduction of sulfonamides. ¹⁸

Vaccines against S. pneumoniae and H. influenzae have dramatically decreased these infections in children. Vaccines given to expectant mothers also provide transplacental antibody to protect newborn children. ^19–21

Low-dose standard IGIM (e.g., 100-200 mg/kg/month) provides some protection to major infection (sepsis, meningitis, pneumonia) but has little effect on decreasing the incidence of otitis, sinusitis, or bronchitis, possibly because insufficient antibody reaches mucosal surfaces. ^22, ²³

Santosham et al ²⁴ prepared an experimental high-titered human IG from donors immunized with pneumococcal, meningococcal and H. influenzae b polysaccharide vaccines (termed bacterial polysaccharide IG). This IG significantly reduced invasive disease due to S. pneumoniae and H. influenzae when given to Apache Indian infants. It also reduced the incidence of pneumococcal otitis media but not the total number of otitis episodes. ²⁵

In larger doses (400 mg/kg), IGIV reduced the frequency of otitis due to pneumococcus in HIV-infected infants ^26, ²⁷ and otitis-prone infants. ^28, ²⁹ Larger monthly doses (750 mg/kg) reduced the number of viral respiratory infections in young infants. ²⁸ Thus, IGIV may be beneficial in recurrent respiratory infections and chronic sinusitis not responding to prophylactic antibiotics, even in patients with no apparent immunodeficient illness. ^29–31

In addition, IGIV has been used in the management of refractory measles and adenoviral pneumonia. ^32, ³³

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Source: https://www.sciencedirect.com/topics/immunology-and-microbiology/primary-and-secondary-antibodies

What Do We Do After the Second Incubation Why Do We Wash the Wells Again

Post-translational Modifications That Modulate Enzyme Activity

3.3.4 Enzyme-linked immunosorbent assay

Early diagnosis with ultrasensitive ELISA

2 Protocol

The Role of Macrophages in Antibody Responses in Vitro

B Advantages and Disadvantages of Tissue Culture Systems

Immunofluorescence

Blocking to Limit Nonspecific Antibody Interactions

Immunocytochemical Analysis of Human Stem Cells

Fc Receptors in Sample

Note

Expansion Microscopy for Cell Biology

3.1.2 Immunofluorescence

The Unfolded Protein Response and Cellular Stress, Part A

5.3.3 General protocol

5.3.3.1 Antigen retrieval techniques

Guide to Protein Purification, 2nd Edition

4.3.4 Antibodies

Methods in Extracellular Matrix Biology

2.5.1 "En bloc" Immunolabeling of Tissue

Passive immunization

Bacterial respiratory infections

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