LRP1 Antibody, FITC conjugated

What is LRP1 and what cellular functions does it serve in research contexts?

LRP1 (Low-density lipoprotein receptor-related protein 1) is an endocytic receptor involved in multiple crucial cellular processes including endocytosis, phagocytosis of apoptotic cells, and cellular lipid homeostasis. It is required for early embryonic development and participates in the plasma clearance of chylomicron remnants and activated LRPAP1. Research has demonstrated that LRP1 modulates cellular events including APP metabolism, kinase-dependent intracellular signaling, neuronal calcium signaling, and neurotransmission. Additionally, it functions as a receptor for Pseudomonas aeruginosa exotoxin A, demonstrating its multifunctional properties in biological systems .

What specific cellular compartments express LRP1 and how can FITC-conjugated antibodies help visualize this distribution?

LRP1 demonstrates a complex distribution pattern across multiple cellular compartments. According to immunohistochemical studies, LRP1 is found in the cytoplasm, nucleus, cell membrane, and extracellular matrix . In the central nervous system, LRP1 is expressed in multiple cell types. Immunofluorescence microscopy studies have shown that LRP1 is expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes . In white matter tracts, LRP1 colocalizes with CNPase-positive areas, indicating expression in oligodendrocyte-rich regions . FITC-conjugated LRP1 antibodies enable visualization of this distribution through applications such as immunofluorescence microscopy, allowing researchers to track LRP1 localization with high sensitivity while simultaneously staining for other markers in multiplexed imaging studies.

What applications are most suitable for LRP1 antibody, FITC conjugated in research settings?

LRP1 antibody, FITC conjugated is versatile for multiple applications in research contexts. According to product specifications, it can be effectively used in:

• Flow cytometry (FCM) - Recommended dilution: 1:20-100

• Immunofluorescence on paraffin-embedded tissues (IF/IHC-P) - Recommended dilution: 1:50-200

• Immunofluorescence on frozen sections (IF/IHC-F) - Recommended dilution: 1:50-200

• Immunocytochemistry (IF/ICC) - Recommended dilution: 1:50-200

These applications make the antibody particularly valuable for studies requiring simultaneous detection of multiple targets, especially in complex tissues like brain sections where LRP1 expression changes are associated with pathological conditions such as experimental autoimmune encephalomyelitis (EAE) .

What is the specificity of the LRP1(Ser4523) antibody and what species reactivity does it demonstrate?

The LRP1(Ser4523) polyclonal antibody specifically recognizes the phosphorylation site at Serine 4523 in human LRP1. This corresponds to phosphorylation sites at serine 4524 in both mouse and rat LRP1 proteins. According to product specifications, the antibody demonstrates confirmed reactivity with human and rat tissues, with predicted reactivity in mouse samples . This specificity is important for phosphorylation-dependent studies, as the antibody was developed using KLH-conjugated synthetic phosphopeptide derived from human LRP1 around the phosphorylation site of Ser4523, making it suitable for studying phosphorylation events that may regulate LRP1 function.

How can LRP1 antibody, FITC conjugated be utilized to study blood-tissue barriers such as the blood-labyrinth barrier (BLB)?

LRP1 has emerged as a potential target for shuttling therapeutics across the blood-labyrinth barrier (BLB), which restricts drug entry into the inner ear tissue. Research has demonstrated that LRP1 is expressed on the BLB, similar to its expression on the blood-brain barrier (BBB) . To study this phenomenon, researchers can employ LRP1 antibody, FITC conjugated in combination with vascular markers to:

• Visualize LRP1 distribution in the cochlea, focusing on barrier-forming regions

• Compare expression patterns between normal and pathological conditions

• Evaluate colocalization with other transporter molecules

For optimal results, thin sections (5-8 μm) of cochlear tissue should be used with the antibody at a 1:50-200 dilution for immunofluorescence applications. In published studies, LRP1 was localized in the basal layers of basilar membrane, Dieters' cells, inner and outer pillar cells, with notably high expression in inner hair cells and hair cell bundles . FITC-conjugated LRP1 antibody allows for simultaneous staining with red-fluorescent markers for vasculature, providing spatial context for receptor localization relative to the BLB.

What methodological approaches are optimal for studying LRP1's role in myelin vesicle (MV) phagocytosis using FITC-conjugated antibodies?

LRP1 has been identified as an essential receptor involved in the phagocytosis of myelin vesicles (MVs). To study this process using FITC-conjugated LRP1 antibodies, researchers should consider the following methodological approach:

• Preparation of FITC-labeled myelin vesicles by isolating myelin from CNS tissue, degrading it, and labeling with FITC

• Cell culture of phagocytic cells (oligodendrocytes, microglia, or astrocytes)

• Incubation of cells with FITC-MVs with or without LRP1 inhibitors (such as GST-RAP)

• Flow cytometric analysis to quantify MV uptake

Control experiments should include:

• Neutralization with GST-RAP to block LRP1 function

• Comparison with LRP1-deficient cells

• Use of MBP-specific antibody to inhibit MV uptake (MBP binding to LRP1 is involved in MV internalization)

This approach allows for quantitative assessment of phagocytosis and can be applied across multiple cell types. Research has demonstrated that when LRP1 function is blocked either through GST-RAP addition or genetic deficiency, MV uptake is significantly inhibited, confirming LRP1's essential role in this process .

How does LRP1 expression change in experimental autoimmune encephalomyelitis (EAE) models, and how can FITC-conjugated antibodies track these changes?

In experimental autoimmune encephalomyelitis (EAE) models, LRP1 expression is significantly upregulated in the central nervous system. Immunoblot analysis has shown substantially increased LRP1 protein levels in both spinal cord and cerebellum extracts from mice immunized with proteolipid protein peptide (PLP) compared to control mice .

To track these changes using FITC-conjugated LRP1 antibodies, researchers should:

• Collect tissue sections from control and EAE model animals at various disease stages

• Perform immunofluorescence microscopy with FITC-conjugated LRP1 antibody (1:50-100 dilution)

• Combine with cell-type specific markers to identify expressing populations:

  • Griffonia simplicifolia isolectin B4 (IsoB4) for microglia

  • GFAP for astrocytes

  • CNPase for oligodendrocytes/white matter

This approach has revealed that in EAE models, LRP1 expression increases in multiple cell types. In normal brain, GFAP-positive astrocytes express LRP1, and this expression is maintained in EAE. White matter tracks in the cerebellum that are CNPase-positive also show strong LRP1 immunoreactivity in both control and EAE conditions . The FITC-conjugated antibody allows for precise localization of LRP1 in tissue sections and facilitates quantitative analysis of expression changes during disease progression.

What are the technical considerations for detecting LRP1 in lymphocytes compared to myeloid cells given their differential expression levels?

Detecting LRP1 in lymphocytes presents unique technical challenges compared to myeloid cells due to significantly lower expression levels. Research indicates that while LRP1 is abundantly expressed on myeloid cells, it is poorly expressed on the surface of lymphocytes . To overcome these challenges when using FITC-conjugated LRP1 antibodies:

• For flow cytometry applications:

  • Use higher antibody concentrations (closer to 1:20 dilution)

  • Increase acquisition time and cell numbers (collect minimum 50,000 events)

  • Include appropriate compensation controls for FITC

  • Consider signal amplification techniques

• For immunofluorescence applications:

  • Use more sensitive detection systems

  • Increase exposure times during imaging

  • Consider tyramide signal amplification to enhance FITC signal

  • Use confocal microscopy for better signal-to-noise ratio

• Sample preparation considerations:

  • Avoid harsh fixation methods that might further reduce detection of low-abundance proteins

  • Optimize permeabilization conditions if detecting intracellular LRP1 pools

  • Consider blocking with both serum and Fc-block to reduce background

This differential expression pattern explains why LRP1's role in lymphocytes has been less thoroughly characterized compared to myeloid cells, where the abundant expression facilitates functional studies .

What sample preparation protocols optimize detection of LRP1 using FITC-conjugated antibodies in different experimental systems?

Optimal sample preparation for LRP1 detection using FITC-conjugated antibodies varies by experimental system:

For Flow Cytometry:

• Harvest cells gently using non-enzymatic cell dissociation solutions when possible

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

• If detecting intracellular LRP1, permeabilize with 0.1% Triton X-100 or commercial permeabilization buffers

• Block with 5% normal serum from the same species as secondary antibody (if using indirect detection)

• Incubate with FITC-conjugated LRP1 antibody at 1:20-100 dilution in blocking buffer

• Wash thoroughly to remove unbound antibody

• Analyze promptly or store protected from light at 4°C

For Immunohistochemistry (Frozen Sections):

• Collect tissue and snap-freeze in OCT compound

• Section at 5-10 μm thickness

• Fix sections briefly (5-10 minutes) with cold acetone or 4% PFA

• Block endogenous peroxidase activity if using enzyme-based detection systems

• Apply FITC-conjugated LRP1 antibody at 1:50-200 dilution

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium to preserve FITC fluorescence

For Paraffin-Embedded Tissues:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin

• Section at 4-6 μm thickness

• Deparaffinize and rehydrate

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Block with 10% normal serum

• Apply FITC-conjugated LRP1 antibody at 1:50-200 dilution

• Use DAPI for nuclear counterstaining

• Mount with anti-fade medium

These protocols should be optimized for specific experimental conditions and cell/tissue types to achieve optimal signal-to-noise ratio.

What controls should be included when using LRP1 antibody, FITC conjugated for experimental validation?

Proper experimental validation requires several critical controls when using LRP1 antibody, FITC conjugated:

Essential Controls:

• Isotype Control: Include a FITC-conjugated non-specific IgG from the same host species (rabbit) at the same concentration to assess non-specific binding.

• Negative Controls:

  • Omission of primary antibody to assess autofluorescence and secondary antibody specificity

  • Use of samples known to be negative for LRP1 expression

  • Application of blocking peptide (the synthetic phosphopeptide used for immunization) to confirm specificity

• Positive Controls:

  • Tissues or cells known to express high levels of LRP1 (e.g., macrophages, astrocytes)

  • Parallel staining with an alternative validated anti-LRP1 antibody targeting a different epitope

• Functional Validation Controls:

  • Compare staining patterns with and without GST-RAP, which blocks LRP1 function

  • For phospho-specific detection, include samples treated with phosphatase to confirm phospho-specificity

• Colocalization Controls:

  • For tissue sections, include co-staining with established markers of LRP1-expressing cells:

    • GFAP for astrocytes

    • CNPase for oligodendrocytes

    • IsoB4 for microglia

• Technical Controls:

  • Single-color controls for compensation in multicolor flow cytometry

  • Fluorescence minus one (FMO) controls to set gating boundaries

  • Serial dilution of antibody to verify optimal concentration

Inclusion of these controls ensures reliable and interpretable results, particularly when studying complex biological systems like the central nervous system or immune cell populations where LRP1 expression varies significantly between cell types.

How can photobleaching of FITC be minimized during long imaging sessions with LRP1 antibody, FITC conjugated?

FITC is susceptible to photobleaching, which can compromise data quality during extended imaging sessions. To minimize photobleaching when using LRP1 antibody, FITC conjugated:

Before Imaging:

• Store antibody solution at -20°C in aliquots to avoid repeated freeze-thaw cycles, which can degrade FITC

• Protect samples from light during all preparation steps including incubation with antibody

• Use anti-fade mounting media containing scavengers of reactive oxygen species (e.g., DABCO, NPG, or commercial anti-fade media)

• Consider using commercially available ProLong™ or SlowFade™ anti-fade formulations

• Allow mounting media to cure completely as per manufacturer's instructions before imaging

During Imaging:

• Use the minimum excitation light intensity necessary for adequate signal detection

• Limit exposure time to the minimum required for sufficient signal-to-noise ratio

• Employ neutral density filters to reduce excitation light intensity

• Use narrow bandpass excitation and emission filters to minimize phototoxicity

• For confocal microscopy:

  • Reduce laser power to minimum effective level

  • Use line scanning rather than point scanning when possible

  • Increase PMT gain rather than laser power when possible

  • Consider resonant scanning for faster acquisition with less damage

• Image regions of interest (ROIs) first before surveying the entire slide

• For time-lapse experiments, reduce the frequency of capture to necessary minimum

Image Analysis Considerations:

• Collect baseline measurements of bleaching rates by repeated imaging of a control field

• Consider mathematical correction for photobleaching in quantitative analyses

• Use ratiometric imaging approaches when possible to normalize for intensity variations

By implementing these strategies, researchers can significantly extend the useful imaging time for FITC-conjugated antibodies and improve data quality in LRP1 localization studies.

What are the optimal storage conditions to maintain the activity of LRP1 antibody, FITC conjugated?

Maintaining the activity of LRP1 antibody, FITC conjugated requires appropriate storage conditions that preserve both the antibody specificity and the fluorophore integrity:

• Temperature:

  • Store at -20°C for long-term storage as specified by manufacturer

  • Avoid storing at 4°C for extended periods as this can lead to gradual loss of FITC fluorescence

  • Do not store at room temperature

• Aliquoting:

  • Upon receipt, divide the antibody into small single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile microcentrifuge tubes for aliquoting

  • Typical aliquot volumes of 10-20 μl are recommended for most applications

• Protection from Light:

  • Store in amber or opaque tubes

  • Wrap storage containers in aluminum foil for additional protection

  • Minimize exposure to light during handling

• Buffer Considerations:

  • The antibody is optimally stored in the manufacturer's buffer containing 0.01M TBS (pH 7.4), 1% BSA, 0.03% Proclin300, and 50% Glycerol

  • Do not dilute the stock antibody unless immediately using

  • If dilution is necessary, use buffers containing stabilizing proteins (e.g., 1% BSA)

• Handling:

  • Allow the antibody to equilibrate to room temperature before opening to prevent condensation

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

  • Handle with clean, powder-free gloves to avoid contamination

• Quality Control:

  • Label all aliquots with antibody name, lot number, date of aliquoting, and expiration date

  • Consider including a positive control experiment periodically to verify antibody performance

  • Monitor for signs of compromised quality (color change, precipitation, or diminished signal)

Following these storage guidelines will help maintain antibody performance throughout its expected shelf life and ensure consistent experimental results.

How is LRP1 expression altered in various disease models, and how can FITC-conjugated antibodies help characterize these changes?

LRP1 expression undergoes significant alterations in multiple disease models, which can be effectively characterized using FITC-conjugated antibodies:

In Experimental Autoimmune Encephalomyelitis (EAE):
LRP1 protein expression is substantially increased in both spinal cord and cerebellum extracts from mice with EAE compared to controls. Immunofluorescence microscopy using FITC-conjugated antibodies reveals increased LRP1 expression in multiple CNS cell types including microglia, astrocytes, and cells within white matter tracts .

In Atherosclerosis:
Studies indicate that decreased LRP1 expression in pro-inflammatory monocytes is linked with atherosclerosis development. LRP1 is implicated in atherogenic plaque formation and the development of the disease. FITC-conjugated antibodies can help track this expression pattern in circulating monocytes and tissue macrophages .

In Vitiligo:
Increased expression of LRP1 is observed in monocytes of vitiligo patients compared to healthy controls. This elevated expression persists even after repigmentation, suggesting LRP1 may play a role in vitiligo progression and represent a potential therapeutic target .

In HIV Infection:
LRP1 is overexpressed in monocytes from patients with long-term, non-progressing HIV infection and from subjects who remain HIV-1-seronegative despite exposure to the virus. This suggests LRP1's potential involvement in protection against HIV-1 infection .

FITC-conjugated LRP1 antibodies facilitate:

• Quantitative analysis of expression changes via flow cytometry

• Spatial characterization of expression patterns using immunofluorescence

• Correlation of expression levels with disease severity and progression

• Identification of cell populations with altered LRP1 expression in heterogeneous samples

These applications make FITC-conjugated LRP1 antibodies valuable tools for understanding the pathophysiological roles of LRP1 in various disease contexts.

What role does LRP1 play in graft versus host disease (GVHD), and how can FITC-conjugated antibodies contribute to this research?

Recent research suggests LRP1 plays a significant role in graft versus host disease (GVHD), with preliminary studies demonstrating that LRP1 deletion in donor murine T cells results in significantly lower GVHD-related mortality in recipient mice with MHC-mismatched hematopoietic stem cell transplantation . This suggests LRP1 may be a potential therapeutic target for GVHD prevention and treatment.

FITC-conjugated LRP1 antibodies can contribute to this research through:

• Characterizing T Cell Populations:

  • Identifying LRP1-expressing T cell subsets via flow cytometry

  • Quantifying changes in LRP1 expression during T cell activation and differentiation

  • Correlating LRP1 expression with T cell effector functions

• Tracking LRP1 in Tissue Infiltration:

  • Visualizing LRP1-expressing T cells infiltrating GVHD target organs

  • Examining spatial relationships between donor T cells and recipient tissues

• Mechanistic Studies:

  • Investigating LRP1-dependent signaling pathways in T cells during GVHD

  • Exploring interactions between LRP1 and other immune modulators

• Therapeutic Development:

  • Screening potential LRP1-targeting compounds for GVHD prevention

  • Monitoring changes in LRP1 expression during therapeutic interventions

Despite the importance of T cells in GVHD development, there is a significant gap in scientific literature regarding LRP1's role in T cell biology . FITC-conjugated antibodies provide a valuable tool to address this knowledge gap and explore LRP1 as a novel target for anti-GVHD treatment strategies.

How can LRP1 antibody, FITC conjugated be used to study LRP1's role as a shuttle across biological barriers?

LRP1 has emerged as a promising target for shuttling therapeutics across biological barriers, including the blood-brain barrier (BBB) and blood-labyrinth barrier (BLB). FITC-conjugated LRP1 antibodies can be instrumental in studying this process:

• Visualizing Barrier Expression:

  • FITC-conjugated antibodies can identify LRP1 expression on barrier-forming cells

  • In the inner ear, LRP1 has been localized in the basal layers of basilar membrane, Dieters' cells, inner and outer pillar cells, and inner hair cells

  • Co-staining with endothelial markers can confirm the presence of LRP1 at barrier interfaces

• Tracking LRP1-Mediated Transport:

  • FITC-labeled LRP1 ligands can be used to monitor transport across barriers

  • For example, Lix-FITC, a reported LRP1 ligand, has been used to study localization in mouse cochleae

  • Time-course studies can be performed to assess the kinetics of transport

• Evaluating Novel Delivery Systems:

  • LRP1-binding peptides (like IETP2) conjugated to therapeutic compounds have been developed to facilitate transport across the BLB

  • FITC-conjugated LRP1 antibodies can help assess binding specificity of these peptides

  • Competitive binding assays can determine the efficiency of novel targeting moieties

• Comparing Different Barriers:

  • FITC-conjugated antibodies enable comparative studies of LRP1 expression and function across different barriers (BBB, BLB, blood-retinal barrier)

  • This can inform tissue-specific targeting strategies

• Assessing Barrier Integrity:

  • Changes in LRP1 distribution can indicate alterations in barrier function

  • FITC-conjugated antibodies can monitor these changes under various pathological conditions

This research has significant therapeutic implications, as demonstrated by the development of LRP1-binding peptides for drug delivery across the BLB to treat inner ear disorders , and similar approaches being explored for CNS drug delivery.

What are the technical considerations for using LRP1 antibody, FITC conjugated in studying phagocytosis of degraded myelin?

LRP1 plays a crucial role in the phagocytosis of degraded myelin, with implications for demyelinating diseases like multiple sclerosis. When using FITC-conjugated LRP1 antibodies to study this process, several technical considerations are important:

• Preparation of Myelin Substrates:

  • Myelin vesicles (MVs) should be prepared from purified myelin using controlled degradation

  • For tracking uptake, MVs can be labeled with fluorophores spectrally distinct from FITC (e.g., Cy3, Cy5)

  • Alternatively, MVs can be labeled with FITC directly if LRP1 antibodies with different fluorophores are used

• Cellular Models:

  • Multiple cell types can be studied including MEFs, oligodendrocytes, microglia, and astrocytes

  • LRP1 function in MV phagocytosis is substantial across these cell types, indicating this activity is not cell-type-specific

  • For microglia, primary cultures or cell lines like BV-2 can be used

• Experimental Controls:

  • GST-RAP should be included to neutralize the ligand-binding activity of LRP1

  • Comparisons between wild-type and LRP1-deficient cells provide definitive evidence

  • MBP-specific antibody can be used to block MV internalization, as MBP binding to LRP1 is involved in this process

• Quantification Methods:

  • Flow cytometry provides quantitative assessment of MV uptake

  • Confocal microscopy allows visualization of internalized vs. surface-bound myelin

  • Z-stack imaging confirms internalization rather than surface adherence

• Co-receptor Considerations:

  • LRP1 may function with co-receptors like calreticulin

  • In oligodendrocytes and microglia, additional LDL receptor homologues that bind RAP (LRP2, VLDL receptor) may contribute

  • Blocking experiments should address these potential interactions

These methodological approaches have established LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors, with important implications for demyelinating disorders and their treatment .

What are common issues encountered when using LRP1 antibody, FITC conjugated, and how can they be resolved?

Researchers may encounter several challenges when working with LRP1 antibody, FITC conjugated. Here are common issues and their solutions:

Low Signal Intensity:

• Possible causes: Insufficient antibody concentration, low target expression, suboptimal fixation, photobleaching

• Solutions:

  • Increase antibody concentration (try 1:20 dilution for low-expressing samples)

  • Extend incubation time (overnight at 4°C)

  • Optimize fixation protocol (mild fixation may preserve epitopes better)

  • Use signal amplification systems (tyramide amplification)

  • Ensure proper storage to maintain FITC activity

High Background:

• Possible causes: Non-specific binding, insufficient blocking, autofluorescence

• Solutions:

  • Increase blocking time and concentration (5-10% normal serum)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Include additional blocking agents (1% BSA, 0.1% fish gelatin)

  • For tissue samples, use autofluorescence quenching reagents

  • Increase wash steps (number and duration)

Poor Specificity:

• Possible causes: Cross-reactivity with related proteins, non-specific binding

• Solutions:

  • Validate with positive and negative controls

  • Include blocking peptide controls

  • Compare with alternative anti-LRP1 antibodies

  • Pre-absorb antibody with non-target tissues/proteins

Inconsistent Results in Flow Cytometry:

• Possible causes: Variability in cell preparation, compensation issues

• Solutions:

  • Standardize cell preparation protocols

  • Use consistent gating strategies based on FMO controls

  • Ensure proper compensation when using multiple fluorophores

  • Acquire sufficient events (minimum 50,000) for low-expressing populations

Reduced Signal in Multiplexed Staining:

• Possible causes: Spectral overlap, antibody competition, steric hindrance

• Solutions:

  • Choose compatible fluorophores with minimal spectral overlap

  • Optimize antibody order and concentration

  • Consider sequential staining protocols

  • Test different fixation and permeabilization conditions

Cross-Species Reactivity Issues:

• Possible causes: Differences in epitope conservation across species

• Solutions:

  • Verify sequence homology of the target phosphorylation site

  • Test the antibody on known positive samples from the species of interest

  • Consider using species-specific antibodies if available

By addressing these common issues systematically, researchers can optimize protocols for specific applications and experimental systems.

How can LRP1 antibody, FITC conjugated be used effectively in multicolor flow cytometry experiments?

Effective use of LRP1 antibody, FITC conjugated in multicolor flow cytometry requires careful experimental design and optimization:

Panel Design Considerations:

• Fluorophore Selection:

  • FITC is excited by the 488 nm laser and emits at ~520 nm

  • Pair with fluorophores that have minimal spectral overlap (e.g., PE-Cy7, APC, BV605)

  • Reserve brighter fluorophores (PE, APC) for low-expression targets

  • Consider the relative expression levels of LRP1 in target populations:

    • Use higher antibody concentrations for lymphocytes (1:20-1:50)

    • Standard concentrations (1:50-1:100) may suffice for myeloid cells

• Titration and Optimization:

  • Titrate the antibody to determine optimal concentration

  • Create a titration series (1:10, 1:20, 1:50, 1:100, 1:200)

  • Select concentration with best signal-to-noise ratio

  • Determine optimal incubation time (typically 15-30 minutes at room temperature)

• Essential Controls:

  • Single-color controls for compensation

  • Fluorescence Minus One (FMO) controls

  • Isotype control (FITC-conjugated rabbit IgG)

  • Biological controls (LRP1-high and LRP1-low expressing cells)

Sample Preparation Protocol:

• Harvest cells gently to preserve surface expression

• Wash twice in cold PBS/2% FBS

• Block Fc receptors (10 minutes, 4°C)

• Add FITC-conjugated LRP1 antibody at optimized concentration

• Incubate 30 minutes at 4°C in dark

• Wash twice with cold PBS/2% FBS

• Fix if necessary (1-2% paraformaldehyde)

• Analyze within 24 hours

Data Acquisition Guidelines:

• Set PMT voltages using unstained controls

• Apply compensation using single-color controls

• Collect sufficient events:

  • Minimum 50,000 events for rare populations

  • 10,000-20,000 events for abundant populations

• Use consistent gates based on FMO controls

• Include time parameter to monitor instrument stability

Analysis Considerations:

• Gate on single cells using FSC-A vs. FSC-H

• Exclude dead cells using viability dye

• Apply consistent gating strategy across samples

• Quantify using median fluorescence intensity (MFI)

• Calculate LRP1 expression relative to isotype control

This approach enables reliable detection of LRP1 across different cell populations and facilitates comparative studies of LRP1 expression in normal and pathological conditions.

By following these guidelines, researchers can effectively incorporate LRP1 antibody, FITC conjugated into multicolor flow cytometry panels for comprehensive immunophenotyping studies.